bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced expression of EcSOD in skeletal muscle blocks chronic heart failure–induced muscle atrophy and exercise intolerance in mice
Authors: Okutsu Mitsuharu , Lira Vitor A. , Zhang Mei , Yan Zhen
Published: June 2021

We have recently identified nitric oxide (NO)‐dependent antioxidant defense system underlying the resistance of oxidative myofibers to catabolic muscle wasting. To further ascertain the function of this defense system in vivo, we subjected mice to dexamethasone (Dex) injection with/without systemic administration of NO donor S‐nitrosoglutathione (GSNO). We show that Dex induced muscle atrophy and increased E3 ligases MAFbx/atrogin‐1 mRNA along with reduction of extracellular superoxide dismutase (EcSOD) protein in glycolytic muscles, which could be prevented by GSNO. Consistent with the hypothesis that EcSOD is important in maintaining muscle mass and function against cachexia, we observed that Dex‐induced muscle atrophy could be significantly attenuated by electric pulse‐mediated gene transfer of EcSOD and in muscle‐specific EcSOD transgenic mice [muscle creatine kinase (MCK)‐EcSOD]. Importantly, when MCK‐EcSOD mice were crossbred with cardiac‐specific calsequstrin [α‐myosin heavy chain (α‐MHC)‐CSQ] mice, a genetic model of chronic heart failure, they presented significant attenuation of catabolic muscle wasting with well‐preserved whole body muscle strength and exercise tolerance. These findings strongly support the functional importance of EcSOD in protecting against cachexia‐induced muscle wasting and loss of function.
A molecular mechanism for frequency dependent skeletal muscle fiber plasticity
Authors: Jaimovich Enrique , Jorquera Gonzalo , Buvinic Sonja , Casas Mariana
Published: June 2021

Frequency of stimulation determines slow or fast fiber type gene expression in muscle.

Fibers isolated from mice flexor digitorium brevis were stimulated at 20 and 90 Hz (270, 0. 3 ms pulses). We analyzed gene expression (qPCR), ATP release (luciferase), IP3 production (ELISA) and calcium signals in Fluo‐4 loaded fibers.

At 20 Hz there was an important, biphasic release of ATP from myofibers which was inhibited by blockers of pannexin channels and dihydropyridine receptors. This stimulation pattern induced a sequential biphasic IP3 increase; mRNA levels for slow troponin I (TnIs) increased and fast troponin I (TnIf) decreased. These changes were inhibited by apyrase, carbenoxolone and IP3R blockers (xestospongin B). We also saw an increase of citrate synthase and a decrease of enolase mRNAs, not inhibited by xestospongin B. At 90 Hz there was no ATP release and IP3 production was much reduced. In these conditions there was an IP3R‐independent increase in TnIf mRNA levels and a decrease in TnIs.

Upon potassium depolarization, IP3‐dependent calcium signals at membrane potential values more negative than those described for calcium release from RyR, associate to an increase in mRNA of the slow isoform of TroponinI.

Our data supports a mechanism involving frequency‐dependent ATP release and activation of P2Y receptors to induce the slow fiber phenotype in skeletal muscle.

FONDECYT 11108467, FONDAP 15010006
Fiber‐type skeletal muscle response to dietary selenium and isoflavone supplementation in male mice
Authors: Stallings Michael T. , Hardman Jeremy M. , Hart Chris M. , Christensen Merrill J. , Hancock Chad R.
Published: June 2021

Previous research has shown selenium (Se) and isoflavone (IF) supplementation influences carbohydrate metabolism and insulin sensitivity. In response to different dietary IF contents and/or supplemental Se, we examined adaptations of mitochondrial proteins and FOXO1a in skeletal muscle. FOXO1a is a transcription factor that suppresses insulin sensitivity and alters glucose metabolism. Male FVB mice were fed diets for six months providing minimal IF or 500 mg/kg equivalents of genistein and daidzein. All other dietary components were equivalent. Selenium was administered by gavage daily (3mg/kg body wt/day as Se‐methylselenocysteine). Results show elevated dietary IF content decreased FOXO1a abundance in Tibialis Anterior (19±4.7%) and Red Quadriceps (RQ) (17±5%) vs. low IF. Supplemental Se decreased RQ FOXO1a levels in low IF mice (59±10% vs. control; p<0.001). Our data show Se also decreases FOXO1a in White Quadriceps (12±4%) and RQ (17±9%) vs. no Se overall. As Se had the greatest effect by decreasing FOXO1a in the RQ, we investigated changes in mitochondrial content by measuring Uncoupling Protein 3 (UCP3) and Cytochrome C (CytC) in the same muscle. Both increased in response to supplemental Se (CytC 126±43%, UCP3 99±31.5% elevated vs. control, p<0.05). These data suggest fiber‐type specific adaptation in response to supplemental Se and elevated dietary isoflavone content.
Translational Supression of Atrogin‐1 and MuRF1 by miR‐23a Integrates Resistance to Skeletal Muscle Atrophy
Authors: Wada Shogo , Kato Yoshio , Ushida Takashi , Akimoto Takayuki
Published: June 2021

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle‐specific ubiquitin ligases, atrogin‐1/MAFbx and muscle RING‐finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy‐associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR‐23a suppresses the translation of both MAFbx/atrogin‐1 and MuRF1 in a 3’ UTR‐dependent manner. Ectopic expression of miR‐23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR‐23a transgenic mice showed resistance against glucocorticoid‐induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.
Melatonin and/or therapeutic exercise induces autophagy‐mediated muscles remodeling in collagenase‐induced osteoarthritic rats
Authors: Hong Yunkyung , Kim Hyunsoo , Lee Seunghoon , Hong Yonggeun
Published: June 2021

Osteoarthritis (OA) is characterized by progressive degeneration of arthritic cartilage, subsequent joint narrowing, and abnormal gait. The purpose of this study is to determine the effects of melatonin and/or exercise on the muscle remodeling and functional gait in collagenase‐induced OA rats. Melatonin (MT) injection was performed twice/day subcutaneously. The treadmill exercise (Ex) was conducted for 1‐hr/day at the frequency of 5 days/week. As results, body weight was significantly reduced by melatonin and/or exercise (P<.05), whereas no significance was shown in gastrocnemius as well as tibialis anterior muscle mass in OA+MT and OA+MT+Ex group at day 28. MAFbx, ubiquitin E3 ligase, was increased in medial gastrocnemius muscle in OA+MT+Ex group at day 7 (P<.05). Intervention also induced an upregulation of autophagic molecules, LC3 and BECN1 (P<.05). At day 28, the relative distribution of MHC1b was significantly increased in gastrocnemius muscle of OA+MT and OA+MT+Ex groups (P<.05). Sarcomeric Nebulin was increased by melatonin and/or treadmill exercise at day 28 (P<.05). Furthermore, as a result of footprint, intervention increased feet contact area. Taken together, melatonin and/or exercise activated autophagic machinery inducing muscle structural remodeling in collagenase‐induced OA rats.
A role for central command in the generation of the exaggerated cardiovascular response to exercise in hypertension
Authors: Liang Nan , Mizuno Masaki , Downey Ryan , Mitchell Jere H , Smith Scott A
Published: June 2021

The cardiovascular response to exercise in hypertension is exaggerated. Evidence suggests that an overactive skeletal muscle exercise pressor reflex (EPR) mediates this circulatory hyperexcitability. Whether other neural inputs likewise contribute to altered hemodynamics in this disease remains unclear. Central command, a neural drive originating in higher brain centers, is known to activate cardiovascular and locomotor control circuits concomitantly. Thus, it is a logical candidate for the production of the abnormal circulatory response to exercise in this disease. Using spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats, we examined whether the cardiovascular response to central command activation is altered in hypertension. Rats were decerebrated and paralyzed. Central command activation was evoked by electrical stimulation of the mesencephalic locomotor region (MLR; i.e. fictive locomotion) with different current intensities (20–50 μA in 10‐μA steps). Blood pressure and heart rate responses increased with each elevation in intensity in both groups. Importantly, the cardiovascular responses to MLR stimulation were larger in SHR compared to WKY at all intensities. These findings suggest that, in addition to the EPR, central command contributes to the generation of the exaggerated cardiovascular response to exercise in hypertension. Supported by NIH HL‐088422
Systemic administration of Interleukin‐1beta enhances thermal prolongation of the laryngeal chemoreflex in decerebrate piglets—implications for Sudden Infant Death Syndrome
Authors: Xia Luxi , Bartlett Donald , Leiter J. C.
Published: June 2021

The laryngeal chemoreflex (LCR) causes apnea and may initiate the process leading to the Sudden Infant Death Syndrome (SIDS). Recent upper respiratory tract infection (URI) and thermal stress are risk factors for SIDS, and elevating body temperature in decerebrate piglets prolongs the duration of LCR apnea. We tested the hypothesis that administering interleukin‐1beta (IL‐1b), a cytokine that is elevated by URIs and in turn elevates body temperature, would enhance thermal prolongation of the LCR. Decerebrate piglets (P4 to P13) were tracheostomized, paralyzed and ventilated, and 0.1 ml of water was introduced into the larynx to elicit the LCR. We recorded the duration of phrenic apnea and respiratory disruption associated with the LCR at normal body temperature and when body temperature was elevated by 2 degrees C. Pretreatment by intraperitoneal (IP) administration of IL‐1b (3 microgm/Kg) enhanced thermal prolongation of the LCR compared to control animals treated simultaneously with 10 mg/Kg IP indomethacin and IL‐1b. We conclude that IL‐1b enhances thermal prolongation of the LCR through a mechanism that requires synthesis of additional inflammatory mediators.

Support: grants 36379 & 42707 from the NICHD
Negative pressure on an occluded limb induces sympathetic activation
Authors: Cui Jian , Blaha Cheryl , Herr Michael D. , Mast Jessica , Muller Matthew D. , Drew Rachel C. , Sinoway Lawrence I.
Published: June 2021

We have shown that venous distension induced by saline infusion into veins of an arterially occluded arm increases muscle sympathetic nerve activity (MSNA) and blood pressure (BP). However, the effects of vascular distension in lower limbs have not been examined. In this study, we employed airtight pressure tanks to apply 100 mmHg negative pressure on an arterially occluded limb (termed “NPOL”) to induce the tissue deformation without fluid translocation to the limb. We hypothesized that the NPOL would induce MSNA activation when applied to upper as well as lower limbs. BP, heart rate, and MSNA from the non‐treated leg were assessed in 13 young healthy subjects with arm or leg NPOL for 2 min. Limb occlusion without suction was used as a control. The NPOL increased limb circumference (arm: 3.8 ± 0.6; leg: 2.8 ± 0.6 %), MSNA (arm: 7.0 ± 1.3 bursts/min; 183 ± 17 units/min; leg: 7.3 ± 1.1 bursts/min; 172 ± 27 units/min) and BP (arm: 4.4 ± 1.0 mmHg; leg: 10.5 ± 3.4 mmHg). It had no effect on heart rate. Occlusion did not evoke responses. These data indicate that the limb tissue deformation evokes MSNA and BP increases. These observations combined with our previous findings suggest that arm and leg vascular distension evokes a powerful pressor reflex. Supported by AHA 0635245 N, NIH P01 HL096570, M01 RR010732 and UL1 RR033184.
Sex‐specific sympathetic responses to chemoreflex stress in healthy young men and women
Authors: Usselman Charlotte Willemina , Nielson Chantelle A. , Luchyshyn Torri A. , Gimon Tamara I. , Shoemaker J. Kevin
Published: June 2021

While muscle sympathetic nerve activity (MSNA) responses to baroreceptor unloading are typically greater in men than women, it is unclear whether chemoreflex responses differ between the sexes. We tested the null hypothesis that MSNA responses to concurrent hypoxia and hypercapnia would not be different between sexes. Microneurographic recordings of fibular nerve MSNA, heart rate (HR) and mean arterial pressure (MAP; Finometer) were acquired from healthy young men and women (n=6 each, 25±3y). Females were tested in the early follicular phase of the menstrual cycle. Following baseline recordings, an oxygen partial pressure of 70 torr and an end tidal carbon dioxide partial pressure of 50 torr were achieved through a rebreathing protocol. Immediately succeeding the rebreathing protocol, an end‐inspiratory apnea commenced following inhalation of these gas concentrations. Baseline values for HR, MAP, and MSNA levels did not differ between the sexes. The HR and MAP responses to chemoreflex stress were also similar between men and women (P>0.05). However, during the apnea increases in MSNA burst frequency (+39±16 versus +23±9 bursts/min, P=0.03) and burst incidence (+54±21 versus +38±15 bursts/100 heart beats, P=0.04) were larger in women than men, respectively. These data suggest that sympathetic responses to an apneic hypoxic‐hypercapnic stimulus are greater in women than in men. Supported by NSERC and CIHR.
Cospeptin, a novel reproductive peptide
Authors: Bryant Chloe , Yosten Gina L.C. , Samson Willis K.
Published: June 2021

Using a bioinformatics screen, our group recently discovered the novel neuropeptide cospeptin. We detected highest levels of cospeptin in the hypothalamus, including the periventricular nucleus, suggesting that cospeptin may influence pituitary hormone secretion. Cospeptin treatment alone failed to alter secretion of anterior pituitary (AP) hormones from dispersed primary rat pituitary cells; however, we found that cospeptin pre‐treatment potentiated release of luteinizing hormone in response to gonadotrophin‐releasing hormone (GnRH) stimulation. To determine if cospeptin plays a role in regulation of reproductive function, we compromised production of endogenous cospeptin by administration of siRNA directed against cospeptin into the lateral cerebroventricle of adult female rats, and used vaginal cytology to track the integrity of the estrous cycle. Treatment with cospeptin siRNA caused a delay in the appearance of the next estrus, and led to a decrease in GnRH receptor message in AP, suggesting cospeptin sensitizes gonadotrophs by upregulating the transcription of the GnRH receptor. These and other results suggest that cospeptin is an important reproductive hormone that modulates the sensitivity of pituitary gonadotrophs to GnRH through regulation of the expression of GnRH receptor.
Role of the sex chromosomal complement (XX or XY) to impact blood pressure and natriuresis in the model of aldosterone escape
Authors: Li Lijun , Tsukerman Susanna , Ji Hong , Sandberg Kathryn , Ecelbarger Carolyn
Published: June 2021

The ability to “escape” by natriuresis from aldosterone infusion coupled to high‐NaCl (HS) diet may be influenced by sex steroids, but also more subtly by the sex chromosomal complement (SCC), i.e., XX or XY. We used a novel transgenic line of mice (four‐core genotypes, FCG) in which gonadal sex (male, M; or female, F) segregates independently from the SCC. FCG mice were bred in house: 1) XX‐F, 2) XY‐F, 3) XX‐M, and 4) XY‐M, and were gonadectomized at 2 months to remove masking effects of sex steroids. Mice (8/genotype) were started on a low‐NaCl (0.085%) diet then implanted with aldosterone‐infusing osmotic minipumps (40 μg/40 g·bw/d). After 3‐days, mice were switched to a HS (5% NaCl) diet. XX mice had significantly higher urine volume, urine sodium, and urine potassium excretion in the HS period, as compared XY. This effect was especially prominent in the XX‐F where sodium excretion on the 3rd day of HS was over 100% higher than in the XY‐M. No differences in food intake were found, although M mice were significantly heavier (about 20–30%). Aldosterone infusion alone caused a modest rise in BP (measured by radiotelemetry) in the XX (5–10 mm Hg), but not the XY. HS plus aldosterone led to a further rise in BP, with M mice appearing more sensitive. Our results may suggest increased sensitivity to aldosterone in the XX versus XY genotypes.
Profile of nitric oxide synthases (NOS), antioxidants, and pro‐oxidant enzymes and oxidative stress (OS) markers in the kidney cortex (KC) during rat pregnancy
Authors: Cunningham Mark W. , Sasser Jennifer , Baylis Chris
Published: June 2021

Normal pregnancy (P) involves increased renal sodium reabsorption, and the resultant increase in renal metabolism could cause increased OS, which could decrease renal nitric oxide (NO) bioavailability leading to suboptimal gestational renal vasodilation. Here, we examined enzymes and markers of NO and OS in early (E), mid‐(M) and late (L) pregnancy (P) (days 3, 12, 20) and post‐partum (PP) rats compared to virgins (V). Protein abundance of endothelial (e) NOS was unchanged and neuronal (n) NOSα fell at LP. In contrast, nNOSβ was elevated at MP, LP, and PP. There were no changes in cytosolic (CuZn) superoxide dismutase (SOD), while mitochondrial (Mn) SOD decreased at EP and extracellular (EC) SOD decreased at MP and LP. Despite reductions in SOD abundance, total antioxidant capacity (TAC) was elevated in EP, MP, and PP. Nitrotyrosine (NT) and the NADPH oxidase subunit p22phox abundance decreased in EP, and NT was low during LP and PP. No changes were seen in KC hydrogen peroxide (H2O2) levels.

Conclusion

The gestational renal vasodilation is known to be NO‐dependent and this study supports a role for increased KC nNOSβ during normal P. Despite increased renal metabolism during P there is no evidence of increased OS since NT levels fall by LP and KC H2O2 levels are unchanged. This may in part be related to an early fall in the p22phox subunit of NADPH oxidase and most likely to the significant increase in KC TAC, which occurs despite reductions in total SOD abundance.

nNOSα
nNOSβ
Nitrotyrosine
p22phox
ecSOD
mnSOD
TAC (mM Trolox/mg)

V
100±15
100±9
100±4
100±9
100±7
100±14
131±9

EP
97±19
99±2
84±6*
66±8*
80±15
44±6*
183±12*

MP
85±14
149±9*
98±9
97±13
68±7*
81±26
191±12*

LP
54±13*
247±43*†
48±4*
93±4
57±9*
79±24
153±11

PP
116±22
138±7*
69±6*
84±3
81±6
113±41
174±10*

p<0.05 vs. V and

p< 0.05 vs. EP.
Aminoimidazole carboxamide ribonucleotide administration attenuates placental‐ischemia‐induced hypertension and angiogenic imbalance in rats
Authors: Banek Christopher T , Bauer Ashley J , Rasmussen Mitchell B , Dreyer Hans C , Gilbert Jeffrey S
Published: June 2021

Previous studies suggest restoration of angiogenic balance can lower blood pressure and improve vascular endothelial cell function in several experimental models of preeclampsia. Stimulation of the adenosine monophosphate kinase (AMPK) pathway has previously been shown to increase vascular endothelial growth factor (VEGF) expression. We hypothesized stimulation of AMPK with aminoimidazole carboxamide ribonucleotide (AICAR) would increase plasma VEGF and attenuate reduced utero‐placental perfusion pressure (RUPP)‐induced hypertension. RUPP was induced on day 14 of gestation (term=21) by introducing silver clips on the inferior abdominal aorta and ovarian arteries. AICAR was administered i.p. (50mg/kg) twice daily from day 14–18, and mean arterial pressure (MAP) and tissues were collected on day 19. Placental phosphorylated‐AMPKα:AMPKα was increased in RUPP+AICAR vs. normal pregnant (NP) and RUPP (0.48±0.06 vs. 0.17±0.01 vs. 0.25±0.06; P<0.05). AICAR also increased plasma VEGF in the RUPP+AICAR vs. RUPP (NP 843±52; RUPP 420±75; NP+AICAR 708±179; RUPP+AICAR 884±128 pg/ml; P<0.05). Lastly, RUPP hypertension was mitigated by AICAR infusion (NP 95±3; RUPP 123±2; NP+AICAR 104±3; RUPP+AICAR 101±5mmHg; P<0.05). These findings suggest pharmacological activation of the AMPK pathway may be useful to restore angiogenic balance and lower blood pressure in hypertensive pregnancies.
Twelve transmembrane helices form the functional core of mammalian MATE1
Authors: Zhang Xiaohong , He Xiao , Baker Joseph , Tama2 Florence , Chang Geoffrey , Wright Stephen
Published: June 2021

MATE1 and MATE2/2‐K, the mammalian organic cation/H+ (OC/H+) exchangers, are key players in renal and hepatic secretion of cationic drugs. They belong to a superfamily of transport proteins (Multidrug And Toxin Extruders) with 1000+ members. The x‐ray structure of the prototypic MATE family member, NorM from Vibrio cholera, shows a protein fold comprised of 12 transmembrane helices (TMHs), as predicted by hydropathy analysis of the majority of MATE transporters. However, the mammalian MATEs are predicted to have a C‐terminal 13th TMH. Here we confirmed the presence of the 13th TMH in mammalian MATE1. Whereas the C‐termini of epitope‐tagged, full length human, rabbit and mouse MATE1 were accessible to antibodies from the extracellular face of the membrane, truncation of these proteins at or near the junction between the 13th TMH and the long cytoplasmic loop that precedes it resulted in proteins that (i) trafficked to the membrane, (ii) had a cytoplasmic C‐terminus, and (iii) supported substrate transport. The latter observation indicates that the functional ‘core structure’ of mammalian MATE consists of the first 12 TMHs, whereas the 13th TMH is not necessary for essential function. Therefore, we used the x‐ray structure of NorM to develop a homology model of the 3D structure of hMATE1 that proved to be stable in Molecular Dynamic simulations when placed (in silico) in a hydrated phospholipid bilayer.
Regulation of ABC transporters by hypoxia in HK‐2 human proximal tubule epithelial cells
Authors: Chung Git , Brown Colin Douglas
Published: June 2021

Hypoxia‐inducible factor‐1 (HIF‐1), a transcription factor that is stabilised during hypoxia, may influence the expression of ABC transporters in the kidney. The aim of this study was to use a proximal tubule epithelial cell line (HK‐2) as a kidney model to determine changes in ABC transporters at an mRNA and functional level when HK‐2 cells were exposed to a hypoxia‐mimetic compound, desferrioxamine (DFO).

Western Blots were used to determine changes in protein levels of HIF‐1 between DFO treated and untreated HK‐2 cells. A qPCR approach was used to determine mRNA expression of ABC transporters in DFO treated and untreated HK‐2 cells. The functional expression of ABC transporters in HK‐2 cells was determined using Hoechst 33342 and CMFDA dye efflux assays.

Western blots of HIF‐1 protein showed significantly increased expression in HK‐2 cells treated with DFO compared with untreated cells. The mRNA expression profiles of MDR1, MRP1 and MRP3 showed an up‐regulation fold change of 1.63, 2.04 and 1.58 respectively in DFO treated HK‐2 cells compared to untreated cells. The magnitudes of the fluorescence of retained Hoechst 33342 and CMFDA dyes were significantly lower in DFO treated HK‐2 cells compared to untreated cells (P<0.01).

These results provide evidence to support the hypothesis that HIF‐1, stabilised with DFO, influences the regulation of ABC transporters in the kidney.
Renomedullary interstitial hyaluronan is important for hydration‐induced diuresis
Authors: Hansell Peter , Palm Fredrik , Stridh Sara
Published: June 2021

Hyaluronan (HA) has been implicated in renal fluid handling due to its water attracting properties and the dynamic regulation of medullary HA levels occurring in response to variations in hydration. To directly test the importance of HA in fluid regulation, HA production was reduced by 4‐methylumbelliferone (4‐MU in drinking water for 5 days) in rats prior to acute hypotonic hydration. Following hydration for 135 min in control rats, diuresis and osmotic excretion increased while sodium excretion and GFR remained unchanged. The medullary HA content was 7.85 ± 1.29 ng/mg protein while that in the cortex was 0.08 ± 0.01 ng/mg protein. In 4‐MU‐treated rats the medullary HA content was only 38% of that in controls (p<0.05) while no change occurred in the cortex. Baseline urine flow prior to hydration was not different from controls. The diuretic response to hydration was, however, only 51% of that in the controls and the osmolar excretion was only 47%. Sodium excretion and GFR were similar to controls and remained unaltered during hydration. To conclude, reduction of renomedullary interstitial HA in rats using 4‐MU reduces the ability of the kidney to respond with an appropriate diuresis upon acute hydration. The results support our previous in vivo and in vitro data and the concept that HA is involved in fluid handling by changing the physicochemical properties of the medullary interstitium. Support: Swedish Science Council.
Urine Concentrating Mechanism: Impact of Vascular and Tubular Architecture and a Proposed Descending Limb Urea‐Na Cotransporter
Authors: Pannabecker Thomas L , Dantzler William H , Layton Anita T
Published: June 2021

We extended a region‐based mathematical model of the renal medulla of the rat kidney, previously developed by us, to represent new anatomic findings on the vascular architecture in the rat inner medulla (IM). The model represents separation of descending vasa recta from the descending limbs of loops of Henle, and a papillary segment of the descending thin limb that is water impermeable and highly urea permeable. Model results suggest that, despite compartmentalization of IM blood flow, IM interstitial fluid composition is substantially more homogeneous compared to the outer medulla (OM). We used the model to study medullary blood flow in antidiuresis and the effects of vascular countercurrent exchange. We also hypothesize that the terminal aquaporin‐1 null segment of the long descending thin limbs expresses a urea‐Na or urea‐Cl cotransporter. As urea diffuses from the urea‐rich papillary interstitium into the descending thin limb luminal fluid, NaCl is secreted via the cotransporter against its concentration gradient. That NaCl is then reabsorbed near the loop bend, raising the interstitial fluid osmolality and promoting water reabsorption from IM collecting ducts. The model predicts that the presence of the urea‐Na or urea‐Cl cotransporter facilitates cycling of NaCl within the IM, and yields a loop‐bend fluid composition consistent with experimental data.
Epigenomic markers for heritable risk of preeclampsia
Authors: Anderson Cindy M. , Wright Michelle L. , Ralph Jody L. , Uthus Eric O. , Ohm Joyce E
Published: June 2021

Preeclampsia (PE) affects 8–10% of women in the US. Long‐term consequences include development of maternal hypertension and hypertension in offspring. The familial risk of PE is well established, though underlying mechanisms are unknown. In this study, our goal was to identify differentially methylated genes common to both maternal peripheral blood cells (MPBCs) and placental tissue of fetal origin in order to determine potential epigenomic markers of heritable PE risk. Methylation quantification of individual CpG dinucleotides in MPBCs and placental tissue from women with and without PE was determined using the Illumina Infinium methylation array (n=6/group). Gain of methylation was seen in 218 CpG dinucleotides from MPBCs, and ~34% of these changes were also present in placenta. Significant methylation loss was observed in 123 CpG dinucleotides, and ~28% of changes were also present in placenta. Validation of methylation was determined by bisulfite sequencing. Differentially methylated loci were primarily located in non‐CpG island regions of genes with known roles in angiogenesis, inflammation and metabolic regulation. This study provides novel evidence for the mechanisms underlying heritable risk for PE, implicating differential transgenerational DNA methylation in key genes associated with the etiology and consequences of PE and suggest that these may serve as novel biomarkers for PE screening.
A prospective, longitudinal study of the improvement of vascular function and cardiac autonomic control following renal transplantation
Authors: Ferrante Kimberly P , Stauss Harald M , Kalil Roberto S
Published: June 2021

Uremic state is strongly associated with autonomic nervous system dysfunction and impaired endothelial vascular function. Furthermore, decreased heart rate variability (HRV) in patients with ESRD is associated with higher rates of cardiovascular events. Therefore, we studied endothelial vascular function and cardiac autonomic control in patients with ESRD before and 3 months, 12 months, and 36 months following renal transplantation (RT). Vascular function was assessed by flow mediated dilatation (FMD) and cardiac autonomic control was assessed by time‐ and frequency‐domain HRV analysis. FMD increased by 50% at one year after RT. One year following RT, time‐ and frequency‐domain HRV parameters tended to increase (e.g., RMSSD 20.7±1.9 ms before RT vs. 25.3±5.4 ms one year after RT), indicating improved autonomic cardiac control. Compared to one year following RT, HRV parameters tended to be slightly lower at 3 years following RT, but were still greater than before RT. Importantly, HRV (SDNN) correlated significantly (R=0.4, P<0.05) with FMD. We conclude that renal transplantation in ESRD patients chronically improves endothelial vascular function and cardiac autonomic control. We speculate that improved endothelial vascular function contributes to reduced cardiovascular events following RT in ESRD patients. Supported by: National Institutes of Health (NHLBI K23 HL080410)
Early cardiovascular alterations induced by zinc deficiency during fetal life and lactation
Authors: Abregu Facundo Mendes Garrido , Sanchez Rodrigo , Veiras Luciana Cecilia , Costa Angeles , Tomat Analia Lorena , Arranz Cristina
Published: June 2021

The aim was to evaluate early cardiovascular alterations in rats exposed to moderate zinc deficiency during fetal life and lactation, and compare the effects in males (m) and females (f).

Wistar rats received low (L,8 ppm) or control (C,30 ppm) zinc diet from pregnancy until offspring weaning (day 21). At 21 days, we evaluate mayor (MM) and minor (mM) myocyte diameter (μm); aortic intima (Ai), media (Am) and perivascular collagen (Ac) thickness (μm); cardiac (C) and aortic (A) nitric oxide synthase (NOS) activity (pmol Cit/g.tissue.min); cardiac thiobarbituric acid substances (Tbars, nmol/mg protein), glutathione (G, mg/g protein), glutathione peroxidase (GPX, μmol/min/mg protein), catalase (CAT, pmol/mg protein) and superoxide dismutase (SOD, U/mg protein) activity in Lm, Lf, Cm and Cf rats.

Cm
Lm
Cf
Lf

MM
14±1
18±1*
15±1
15±1

mM
8,6±0,5
10,7±0,3*
9,4±0,5
10,1±0,3

Ai
23,3±0,2
18,3±0,1*
23,9±0,2
18,9±0,2†

Am
280±2
298±2
287±3
280±2

Ac
16±1
24±1*
12±1
23±2†

C NOS
249±5
205±5*
247±3
201±3†

A NOS
225±5
172±5*
211±6
179±6†

G
9±1
3±1*
10±1
6±1†

GPX
81±8
65±6*
85±6
68±8†

Tbars
0.41±0.05
0.32±0.02
0.22±0.04*
0.18±0.02

No differences were observed in cardiac SOD and CAT activity p<0.01:*vs Cm;†vs Cf; N=6/group

These early cardiovascular alterations may participate in the programming of adult diseases

Supported by University of Buenos Aires, CONICET, ANCyT, Argentina
Potassium Adaptation Is Impaired in Hypophysectomized Rats
Authors: Youn Jang H. , Oh Young Taek
Published: June 2021

K+ homeostasis is critical for normal cardiovascular and neuromuscular function. The kidneys have a remarkable capacity to regulate K+ excretion to match K+ intake. This so‐called “K+ adaptation” is critical for chronic K+ balance. We recently showed that K+ adaptation can occur without altering plasma K+ or aldosterone level and that gut sensing of K+ intake enhances renal K+ excretion independent of plasma K+ or aldosterone. We propose that K+ adaptation is mediated by an afferent neural pathway from the gut and secretion of a humoral factor(s) by the pituitary. To test this hypothesis, we examined whether K+ adaptation is impaired in hypophysectomized (HS) rats. Normal and HS rats (n=4 each) were initially fed on a control diet (1% K+) for 3 days and subsequently on a low K+ diet (0.33% K+). Animals were individually housed and had free access to food only at night (6 PM–8 AM). Renal K+ excretion was determined for the absorptive (6 PM–8 AM) and the postabsorptive (8 AM–6 PM) periods. In normal rats, reducing K+ intake to 1/3 of control profoundly decreased both absorptive and postabsorptive K+ excretion. In HS rats, while absorptive K+ excretion decreased similarly, postabsorptive K+ excretion decreased in time courses much slower than those in normal rats (Figure). In conclusion, K+ adaptation was impaired in HS rats, supporting the hypothesis that the pituitary is involved in K+ adaptation. (supported by NIDDK)
Chronic estrogen and 5‐HT1A agonist treatment reduce sodium appetite in ovariectomized rats
Authors: Melo Roberto Laureano , Fonseca Fabricia Viana , Araujo Iracema Gomes , Silva-Almeida Claudio , Marcacini Victor Araujo , Mecawi Andre Souza , Reis Luis Carlos
Published: June 2021

We have shown that ovarariectomized (OVX) rats exhibited higher salt intake compared to estrogen‐replaced rats (OVX+E2) particularly in models of induction of sodium appetite dependent on angiotensin II and low sodium plasma levels. This result was ascribed to enhanced AT1 receptors activity. In male rats we reported that repeated 5‐HT1A agonist administration reduced sodium appetite response related to somatodendritic 5‐HT1A autoreceptor desensitization. Therefore, we investigate the effect of systemic acute and chronic 8‐OH‐DPAT administration on the sodium appetite in normally hydrated or sodium‐depleted Wistar rats (furosemide, 20mg/kg, sc, 24h before experiment) in OVX+oil and OVX estrogen replaced (OVX+E2, estradiol cypionate, 10μg/animal) rats. Acute 8‐OH‐DPAT (250μg/kg, ip, during 7 days) induced an increase of 0.3M NaCl intake. OVX+E2 rats were not responsive to 5‐HT1A agonist in basal condition. Chronic administration of 8‐OH‐DPAT reduced the saline intake compared to OVX+vehicle, control group (2.3±0.17ml/100g vs 4.2±0.2ml/100g, at 120 min after fluids presentation) in sodium‐depleted rats. E2 replacement strengthened the antinatriorexigenic response of OVX+8‐OH‐DPAT treated rats (1.7±0.1ml/100g vs 4.2ml/100g, at 120 min after fluids presentation). Results suggest that chronic administration of 5‐HT1A agonist associated with E2 replacement therapy differentially trigger mechanisms for neural signaling to sodium satiety, possibly through recruitment of serotonergic pathways in the dorsal raphe nucleus.

Support: FAPERJ & CNPq
Vasopressin Not Aldosterone is associated with Changes in Body Mass and Urine Volume during a Controlled 4 d Fluid Intake Intervention
Authors: Johnson Evan C. , Munoz Colleen X , LeBellego Laurent , Klein Alexis , Jimenez Lilianna , Kupchak Brian R , Kraemer William J , Casa Douglas J , Maresh Carl M , Armstrong Lawrence E
Published: June 2021

Arginine vasopressin (AVP) and aldosterone (ALDO) act on renal nephrons to conserve water and sodium in response to decreased total body water, across a wide range of human 24‐h fluid intakes (FI). It is unknown if resting values of AVP or ALDO differ between habitual high FI (HIGH) and low FI (LOW) women during daily activities, and how these groups respond to FI modification. Therefore, the diet records of 120 women were analyzed to identify 14 HIGH (3.34 ± 0.56 L/d) and 14 LOW (1.62 ± 0.48 L/d). Next, the FI of HIGH was reduced (2.00 ± 0.21 L/d) and LOW was increased (3.50 ± 0.13 L/d) over 4d via water prescription. Urinary and blood variables were measured each morning. No main effects of treatment were observed for body mass or ALDO. However, significant interactions, time X treatment, were observed for AVP (F[2, 22] = 40.16, p<.001), and urine volume (F[2, 26]=46.15, p<.001). Post‐hoc tests showed a significant difference in AVP; (F[1, 11] = 83.32, p< .001) from baseline (3.93 ± 1.34, 8.99 ± 2.69 pg/mL) to treatment (8.24 ± 3.22, 4.82±1.08 pg/mL) and in urine volume F(1, 13) = 61.95, p<.001 from baseline (1.90 ± 0.56, 0.79±0.26 L/d) to treatment (1.24 ± 0.22, 2.23 ± 0.44 L/d) between HIGH and LOW respectively. These results demonstrate that changes in AVP, not ALDO, (a) provide the primary defense of total body water, and (b) distinguish small changes (1.1 – 1.5 L/d) in daily FI. Supported by Danone Research, France.
Regulation of TRPV2 in Magnocellular Neurons of the Supraoptic Nucleus in Rat
Authors: Nedungadi Thekkethil Prashant , Walch Joseph D , Cunningham J. Thomas
Published: June 2021

Transient receptor potential vanilloid channel 2 (TRPV2) is activated by hypoosmolality in expression systems. Our objective was to study the role TRPV2 in the magnocellular cells (MNC) of the supraoptic nucleus (SON) in a bile duct ligated rat (BDL) model of hepatic cirrhosis associated with inappropriate vasopressin (AVP) release. We hypothesized that changes in TRPV2 channel expression in AVP, but not oxytocin (OXY) neurons, may contribute to inappropriate AVP release in BDL rats. We used laser capture microdissection (LCM) to capture 7–10 MNC AVP neurons for real‐time RT‐PCR to measure TRPV2 mRNA. The 2−ΔΔCt for TRPV2 mRNA in the LCM AVP neurons of SON in BDL rats averaged 2.5 ± 0.5 vs. 1.0 ± 0.3 in shams. Western blot analysis indicated that TRPV2 protein also was increased in SON by BDL. Following BDL, TRPV2 immunostaining and TRPV2/AVP colocalization was significantly increased in the SON (SHAM TRPV2, 41 ± 2; BDL TRPV2, 52 ± 4; SHAM AVP/TRPV2, 23 ± 1; BDL AVP/TRPV2, 35 ± 2). However, TRPV2 and OXY colocalization was not affected (SHAM OXY/TRPV2, 12 ± 1; BDL OXY/TRPV2, 8.75 ± 1.3). Increased TRPV2 mRNA and protein expression in AVP SON neurons of BDL rats could contribute to changes in osmosensitivity related to inappropriate AVP release. R01 HL062579.
Expression of intestinal mucins may be affected by the dietary protein source
Authors: Onyango Edward M , Habiyambere Bupe M
Published: June 2021

Mucin glycoproteins cover epithelial surfaces of ducts, including the digestive tract, in the body. The study investigated the effect of dietary protein on the expression of mucins in intestinal epithelia of mice. Male weanling mice were randomized into 4 groups of 8 mice each. The source of protein (plant or animal origin) and the level of each protein (10% or 20% crude protein) were arranged in a factorial manner to produce 4 diets which were randomly assigned to the four groups of mice. Mice were provided the diets for 14 days and euthanized on the fifteenth day. Mucosa and histological sections were prepared from the jejunum and colon of the intestines. Total RNA was prepared from the mucosa using Trizol®. cDNA was prepared using random hexamers and specific primers were used for real time PCR amplification of the message for Muc2 and Muc3. Expression of Muc2 in the jejunum was higher (1.45 fold) in mice fed plant protein compared to those fed animal protein. There was no difference in expression of Muc2 in the colon with the different protein sources. Expression of Muc2 did not vary with the level of dietary protein in both the jejunum and colon. Expression of Muc3 in the jejunum and colon did not vary with the level or source of dietary protein. It is concluded that expression of intestinal mucins may be affected by the source of protein in the diet.

Research was supported by a RDC grant from East Tennessee State University.
Leptin receptor deficiency causes intestinal hyperplasia and altered membrane abundance of glucose transporters
Authors: Dominguez Jessica A , Rieg Timo
Published: June 2021

Increasing evidence supports an important role for the small intestine in the control of glucose homeostasis and the pathogenesis of type 2 diabetes. We studied morphological changes in the small intestine and expression of the Na+/glucose transporter 1 (SLC5A1 or SGLT1) and glucose transporter 2 (SLC2A2 or GLUT2) in leptin receptor deficient (db/db; n=6) and control (db/con; n=10) mice. db/db mice were obese (47±1 vs 25±1 g; P<0.01), hyperglycemic (499±43 vs 126±5 mg/dl; P<0.01) and exhibited small intestinal hyperplasia vs db/con as determined by measuring total intestinal length (36.8±1.5 vs 30.0±0.7 cm; P<0.01). Moreover, db/db mice showed increased mucosal mass, including longer villi (673.0±14.3 vs 483.3±19.9 μm; P<0.01) and deeper crypts (112.0±1.7 vs 97.7±3.1 μm; P<0.01). Intestinal proliferation was quantified by staining for proliferating cell nuclear antigen (PCNA). Proliferation was higher in db/db vs db/con (1760±93 vs 1037±28 PCNA‐positive cells/100 crypts; P<0.01). Immunohistochemistry revealed decreased apical membrane abundance of SGLT1, but, in contrast, increased apical membrane abundance of GLUT2 in db/db vs. db/con. These findings suggest that changes in SGLT1 and GLUT2 are present under type 2 diabetic conditions and might contribute to a dysbalance in intestinal homeostasis ultimately leading to intestinal hyperplasia, a situation comparable to diabetic renal hypertrophy.
AQP9 and UT‐A facilitate hepatocyte basolateral membrane urea permeability in mouse
Authors: Jelen Sabina Katarzyna , Gena Patrizia , Lebeck Janne , Prætorius Jeppe , Fenton Robert , Frøkiaer Jørgen , Nielsen Søren , Calamita Giuseppe , Rützler Michael
Published: June 2021

In mammals, nitrogen from protein degradation is disposed as urea. Several studies have investigated expression of urea transporters in hepatocytes, where urea is produced. Nevertheless, the role of protein facilitated urea transport in liver remains unknown. In the presented study we determine urea permeability in isolated basolateral hepatocyte membranes by stopped flow light scattering measurements. The urea channel inhibitors phloretin and dimethylurea reduced the urea permeability by 70% and 40%, respectively. In basolateral membranes isolated from AQP9 (AQP9−/−) and UT‐A (UT‐A−/−) single knockout, and AQP9−/−:UTA−/− double knockout mice, urea permeability (Purea) was decreased by 30%, 40%, and 76%, respectively, compared to wild type mice. In addition, we investigated the physiological role of AQP9 in mouse hepatocyte function following exposure to high oral glutamine doses or high protein diet. These conditions did not affect the concentrations of urea or ammonia in AQP9−/− mouse tissues and plasma, respectively. Similarly, hepatocyte urea synthesis of UT‐A−/− mice appeared normal. We currently conclude that both, AQP9 and a UT‐A gene product, constitute primary but redundant urea channels in hepatocytes.

This study was supported by the Lundbeck Foundation.
Regulation of keratin intermediate filaments by lysine acetylation
Authors: Snider Natasha Tasheva , Leonard Jessica M , Kwan Raymond , Omary M. Bishr
Published: June 2021

Lysine acetylation and sumoylation are ubiquitous evolutionarily‐conserved posttranslational protein modifications that regulate important cellular functions, including cytoskeleton dynamics. Intermediate filaments are one of three major components of the cytoskeleton and are known to be extensively regulated at the posttranslational level. The aim of this study was to determine the role of lysine acetylation on the regulation of the cytoplasmic intermediate filament keratin 8 (K8). We used available proteomic data and site‐directed mutagenesis to identify Lys‐207 as a major acetylation site on human K8. Wild type K8 acetylation is increased in response to high glucose. The cytoplasmic deacetylase SIRT2 directly associates with K8 under low, but not high, glucose conditions. Analysis of liver tissues from mice with chronic liver injury induced by 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) show reciprocity of K8 acetylation and sumoylation. Our data demonstrate that keratin intermediate filaments are dynamically regulated by lysine acetylation and sumoylation in response to cellular energy status and tissue injury. This study was supported by NIH grants DK52951 (to M.B.O.) and DK93776 (to N.T.S).
Direct uptake of copper from ceruloplasmin by cells with and without CTR1, and potential involvement of a copper reductase
Authors: Ishida Michael , Ramos Danny , Mar David , Lee Kyoung Jin , Linder Maria C
Published: June 2021

The blood plasma protein, ceruloplasmin (Cp), is well known for its role in enhancing efflux of iron from certain cells; but it also has other functions, and its copper enters cells all over the mammalian organism, as shown by i.v. infusion of 67Cu‐labeled Cp into rats. In current studies, the ability of Cp to directly donate its Cu to cultured cells was investigated. 64Cu‐labeled human Cp secreted by HepG2 cells was purified and incubated in serum‐free medium with polarized human mammary epithelial cell monolayers with tight junctions (PMC42 cells), applied to the basolateral (“blood” side). Most of the radioactivity entered the cells and 30–50% of what entered was released into the apical (“milk”) fluid. Larger amounts of 64Cu‐Cp were produced in mice and purified for incubation with mouse embryonic fibroblasts (MEF) that did and did not express Ctr1 (kindly provided by Dennis Thiele, Duke University). 64Cu from Cp accumulated in both types of MEFs at a linear rate over 3 h, and was internalized, most being present in the cytosol. Uptake was somewhat less in Ctr1−/− MEFs. Uptake of Cp‐64Cu (which is not exchangeable) was inhibited by an excess of Cu(I) and Cu(II)‐histidine; and the presence of external Cu(II) reductase activity was verified. We conclude that Ctr1 and another (unknown) transporter take up Cu from Cp, and that this is mediated by a Cu reductase. Supported in part by US PHS Grant HD46949.
A2B Receptors on Hematopoietic Cells Control Accumulation of Dendritic Cells after Myocardial Infarction
Authors: Ryzhov Sergey , Yuryeva Ksenia , Novitskiy Sergey , Biktasova Asel , Dikov Mikhail M , Biaggioni Italo , Feoktistov Igor
Published: June 2021

Dendritic cells (DC) can initiate immune reactions contributing to myocardial damage and remodeling after acute myocardial infarction (AMI). Interstitial adenosine is elevated in ischemia. We have previously shown that A2B adenosine receptors attenuate DC generation from myeloid precursors. In this study we tested the hypothesis that A2B adenosine receptors on hematopoietic cells control DC accumulation after AMI. After lethal irradiation of wild type (WT) mice, we performed bone marrow transplantations from A2B receptor knockout (KO) and from WT mice to create A2BKO‐WT chimeras and WT‐WT control, respectively. After confirming that A2BKO‐WT had greater than 90% of the hematopoietic cells replaced by week 8, AMI was created by permanent ligation of the left anterior descending coronary artery. Flow cytometry analysis on day 7 after AMI revealed significantly greater accumulation of cardiac CD11c+MHCIIhigh DC in the hearts of A2BKO‐WT chimeric mice compared with WT‐WT animals (35.0 ± 4.7 and 18.3 ± 2.1 × 103 cell/heart, respectively, p<0.05). The number of DC in the hearts of sham‐operated A2BKO‐WT and WT‐WT mice was virtually the same (2.5 ± 1.7 and 3.2 ± 1.0 × 103 cell/heart, respectively). We conclude that activation of A2B adenosine receptors on hematopoietic cells significantly attenuates AMI‐induced accumulation of CD11c+MHCIIhigh DC in the heart. Support: R01HL095787 and R01CA138923.