bluuebunny/crossref_metadata_2025 huggingface dataset:
Intrarenal Blockade of Angiotensin II AT1 Receptor Abolishes Renal Cortical Hypoxia in Salt Restricted Animals
Authors: Patinha Daniela , Pihl Liselotte , Palm Fredrik
Published: June 2021

Low salt (LS) diet results in activation of the renin‐angiotensin system (RAS) to maintain volemia and arterial blood pressure. We hypothesized that LS‐induced RAS activation increases Na+ transport in the kidney contributing to hypoxia also in the otherwise healthy kidney since renal blood flow (RBF) is not under metabolic control.

Male Sprague Dawley rats were fed normal (NS, 0.4%) or low (LS, 0.01%) salt diet during two weeks. Thereafter, catheters were placed in the carotid artery and femoral vein for blood pressure measurement and infusion of saline containing 3H‐inulin, respectively, in anesthetized (thiobutabarbital) animals. The left kidney was immobilized and a catheter was placed in the renal artery. Measurements were obtained during a baseline and after infusion of the angiotensin II AT1‐receptor blocker candesartan (4.2 µg/kg) directly into the renal artery. Oxygen consumption (QO2) was estimated from the arterio‐venous difference in O2 content multiplied by RBF (Transonic flow probe). Renal cortical and medullary partial pressures of oxygen (PO2) were measured using Clark‐type electrodes. Mean arterial blood pressure, glomerular filtration rate and RBF were similar in both groups. The LS rats had less efficient Na+ transport along with higher QO2 compared to NS. LS reduced cortical PO2 whereas medullary PO2 was increased compared to NS. Importantly, acute AT1‐receptor inhibition by candesartan normalized cortical PO2 and restored the cortico‐medullary oxygen gradient in the animal receiving LS.

These results indicate that LS‐induced cortical hypoxia is associated with angiotensin II AT1 receptor activation, which induces hypoxia in the kidney cortex.
Coronary Ischemia Reperfusion (I/R) Increases Degranulation of Cardiac Mast Cells (CMC) Similarly in Male and Female Rats 8 Weeks After Ischemia
Authors: Scrogin Karie , Tolia Sanika , Samarel Allen
Published: June 2021

Perimenopausal women have a higher risk of mortality within 2 years of myocardial infarction than similarly aged men despite having less left ventricular dysfunction. Cardiac mast cells (CMC) contribute to inflammation after I/R injury. Recent work suggests that estrogen stimulates mast cell degranulation. We tested whether I/R produced increases in CMC numbers and whether degranulation was more extensive 8 weeks after I/R in male or female rats. The number of stable or degranulating CMCs was determined in regions high in CMC density. Three regions were studied: 1) the ventral epicardial surface at the base of the superior vena cava, 2) the rostral pole of the left atrium and 3) the dorsal surface surrounding the right and left pulmonary veins. Both males (P<0.001) and females (P<0.05) showed reductions in fractional shortening (FS) after I/R compared to controls, while males had lower FS after I/R than females (19.3 ± 2.5 vs. 33.3 ± 3.4%, P<0.01). Left ventricular end‐diastolic diameter was increased after I/R only in males (0.77± 0.08 vs. 0.99 ± 0.04, P<0.01). Numbers of CMCs of either sex was not different 8 weeks after I/R in any region examined. The number of degranulating CMCs and the percent of total CMCs that were degranulating were increased in region 3 after I/R in both sexes (26.8 vs. 77.8% in males, P<0.001; and 25.8 vs. 72.3% in females, P<0.001). The percentage of degranulating CMCs was increased similarly across sex in regions 1 and 2 (45‐59% vs. 23‐29%, P<0.05). The data indicate that CMC degranulation continues 8 weeks after I/R injury in regions rostral to the original ischemia. This effect is similar in male and female rats despite less I/R‐induced left ventricular dysfunction in females.
Renal Oxidation of ESS diabetic rats is minimized by EPA
Authors: Osiecki Natalia , Diaz Tomás , Ruiz Pecchio Adriana , Eynard Aldo , Repossi Gastón , García Néstor
Published: June 2021

Salt sensitivity (SS) is associated with increased cardiovascular risk in diabetic patients. This abnormal mechanism results in an increased renal oxydation and decreased urinary sodium excretion and if it is maintained over time can develop hypertension and its complications. Currently the Western diet has limited content of polyunsaturated fatty acids, with antioxidant capacity such as ecoisapentaneoico acid (EPA). Therefore we hypothesized that nutritional supplementation with EPA, prevents SS in DM rats by decreasing renal oxidative stress. Our objectives were; to asses SS of eSS diabetic rats and to investigate the ability of EPA to prevent SS in these rats.

Methods

Wistar rats were used as healthy controls. Type II diabetic rat group (eSS), 3 months old, were divided in 3 groups, diabetic control (eSS), eSS treated with arachidonic acid (pro‐oxidant)(2.5mg / ip, per month) (eSS + AA) and eSS treated with EPA (2.5mg / ip per month) (eSS + EPA). Animals were treated during 1 year, then placed in metabolic cages and subsequently underwent 2 experimental periods of 7 days each with normal sodium diet (0.4% NaCl)(NNaD) and high salt diet (4% NaCl)(HNaD). At the end of each period weight, systolic blood pressure (SBP), HbA1c, triglycerides (Trig), cholesterol (Chol), creatinine (Cre), kidney γ‐glutamyl transpeptidase activity (γGTP)​​, urinary protein excretion (UprotV).

Results

eSS rats had elevated HbA1c, Tri, Chol and reduced body weight vs. Wistar control group. Renal function was normal during the experimental period. The eSS + AA group also showed elevation of HbA1c, Trig, with no change in Cre and Chol. During NNaD SBP was 119 ± 3 mmHg and after HNaD 125 ± 1 mmHg (p <0.05). In contrast, EPA + eSS lowered HbA1c (5.6 ± 0.3% vs 7.0 ± 0.2%, p <0.05), Trig (175±1 mg/dl vs 245±12 mg/dl p <0.05), and increased body weight (424±11 g vs 511±22gr, p <0.05) vs eSS. EPA supplement prevented the increase of SBP during the HNaD (126±2 mmHg vs. 128±2 mmHg p> 0.5). eSS + AA did not differ from eSS group. No significant difference was observed in UprotV between groups. Urinary sodium excretion increased with HNAD. Renal interstitium γ‐GT activity in eSS group was 0.65±0.04 au and 0.61±0.02 au in eSS + AA group, while in EPA + eSS 0.5±0.03 (p <0.05).

Conclusion

The salt sentitive eSS rats have hypertriglyceridemia and elevated HbA1c. Nutritional supplementation with EPA prevented the salt sensitivity, the increased of HbA1c and triglycerides. These changes were associated with lower kidney γ‐GT ​​activity, suggesting that the EPA dietary supplement can be used in diabetic patients to prevent salt sensitivity and improve metabolic abnormalities.
Identification of CNS Sites Involved in the Cardiovascular and Renal Responses Elicited by Nociceptin/Orphanin FQ in Conscious Hypertensive Rats via c‐Fos Immunocytochemistry.
Authors: Hollister Bernadette , Nguyen Tin , Franklin Cynthia , Rangel Yolanda , Gottlieb Helmut
Published: June 2021

Intracerebroventricular (ICV) administration of the opioid‐like peptide agonist, nociceptin/Orphanin FQ (N/OFQ), produces a free water diuresis in addition to marked depressor effects on cardiovascular function and renal sympathetic nerve activity (RSNA). This study examined the changes in cFos expression in forebrain regions of angiotensin II high salt diet (ANGII) hypertensive rats following ICV administration of N/OFQ or saline vehicle. Protocol: 14 days prior to the experiment day, rats were implanted with an osmotic pump filled with angiotensin II and placed on a 2% high salt diet. At the day of the experiments, animals were placed in a metabolic cage and allowed to habituate for 3 hours. Following habituation, then two 15 minutes control periods, rats were than microinjected with either saline vehicle or nociceptin. Ninety minutes after the ICV injection, animals were perfused. Alternate sets of forebrain sections were processed for cFos using a commercially available antibody (Oncogene AB‐5). Results: ANGII treatment significantly increased cFos staining in the magnocellular and parvocellular PVN, SON, PNZ, OVLT, MPA and DMH brain sites as compared to Sham rats. ICV N/OFQ produced significant decrease in c‐Fos staining in the magnocellular PVN, SON, PNZ, OVLT, MPA and DMH of ANGII rats as compared to ANGII group microinjected with saline vehicle. Together, central N/OFQ receptor activation in these forebrain regions may contribute directly to or indirectly participate in the neuropathways involved in the cardiovascular and renal effects of N/OFQ. Supported by NIH 5SC2 HL104639
Angiotensin II Reduces mRNA Expression for Glutamate Transporters and Glutamine Synthetase in Cultured Hypothalamic Astrocytes
Authors: Ruginsk Silvia , Vechiato Fernanda , Elias Lucila , Antunes‐Rodrigues José , Cruz Josiane
Published: June 2021

We have previously shown that ANG‐II decreases glutamate uptake in hypothalamic cultured astrocytes (Cruz et al. Faseb Journal 2013). We hypothesized that ANG‐II may modulate extracellular glutamate availability through a direct effect on glutamate transporters (GLT‐1 and GLAST), as well as on glutamine synthetase (GS) expression by these cells. Therefore, in the present study we investigated the role of ANG‐II on GLT‐1, GLAST and GS mRNA expression in hypothalamic astrocyte cell cultures. For this purpose, cultured astrocytes were stimulated with ANG‐II (100 nM) during 30 or 60 minutes and mRNA expression was evaluated by Real Time PCR. The results showed that 30 minutes of stimulation with ANG‐II induced a significant decrease (N=3‐4 per group) in mRNA expression for GLT1 ((1±0.01 vs. 0.8±0.04 fold, p<0.01), GLAST (1±0.01 vs. 0.8±0.03 fold, p<0.001) and GS (1±0.02 vs. 0.8±0.01 fold, p<0.001) in astrocytes. After 60 minutes, however, the expression of all analyzed transcripts had returned to basal levels. In conclusion, ANG‐II can induce a significant decrease in glutamate transporters and GS mRNA expression in hypothalamic astrocytes, suggesting that high extracellular levels of ANG‐II may produce an increased extracellular glutamate availability in the hypothalamus, which could contribute to an enhanced magnocellular neuronal excitability. Financial Support: FAPESP, CNPq, CAPES.
The disruption of KCNJ10 stimulates the expression of Na‐activated K channel in the basolateral membrane of the thick ascending limb
Authors: Zhang Chengbiao , Wang Lijun , Su Xiao‐tong , Lin Dao‐hong , Wang Wen‐hui
Published: June 2021

Previous study has demonstrated that the disruption of Kcnj10 decreased the expression of NCC in the distal convoluted tubule (DCT). The aim of the present study is to examine the role of Kcnj10 in contributing to the basolateral K conductance in the cortical thick ascending limb (cTAL) using Kcnj10+/+ and ‐/‐ mice. The patch‐clamp studies detected a 40 pS and a 80 pS K channel in the basolateral membrane of the cTAL. Moreover, the probability of finding the 40 pS K was significantly higher in the upper part of the cTAL than in the lower portion. Immunostaining showed that Kcnj10 was expressed mainly in the basolateral membrane of the cTAL close to glomerulus but not in all portion of cTAL. The disruption of Kcnj10 completely eliminated the 40 pS K channel but not the 80 pS K channel, suggesting the role of Kcnj10 in forming the 40 pS K channel of the cTAL. Also, the disruption of Kcnj10 increased the probability of finding the 80 pS K channel in the cTAL. Because the channel open probability of the 80 pS K channel in the KO mice was similar to those of WT mice, this suggests that the disruption of Kcnj10‐induced increase in the 80 pS K channel is mainly achieved by increasing K channel number in the plasma membrane. The whole cell recording further showed that K reversal potential measured with 5 mM K in the bath and 140 mM K in the pipette was the same in the WT and KO mice. Moreover, Western blot and immunostaining show that the disruption of Kcnj10 did not affect the expression of NKCC2. We conclude that KCNJ10 is expressed in the basolateral membrane of cTAL and that the disruption of Kcnj10 has no significant effect on the membrane potential of the TAL and NKCC2 expression.
Intestinal Phosphate Regulation in Chronic Kidney Disease.
Authors: Hogg‐Cornejo Veronica , Sutherland Eileen , Dobrinskikh Evgenia , Levi Moshe
Published: June 2021

Increase in serum phosphate (Pi) level (hyperphosphatemia) is a common clinical problem in chronic kidney disease (CKD). Systemic Pi homeostasis is balanced through three major mechanisms: intestinal uptake, retention or release from bone, and renal reabsorption. Sodium‐phosphate co‐transporter type 2b (NaPi2b) is a major phosphate transporter in the enterocytes of the small intestine. Alport syndrome is a genetic disorder characterized by CKD and hearing loss. We have studied Alport CKD model in mice that have a mutation in a COL4A3 gene on an SvEv129 background. Alport mice (males) have less body (19.6 ± 1.4 g compared to 25.6 ± 0.8 g in WT mice) and kidney weight (177.8 ± 10.4 mg compared to 216.8 ± 16.3 mg in WT mice). By the 9‐10 weeks of age Alport mice develop signs of CKD and hyperphosphatemia: they have increased level of the serum blood urea nitrogen (47.43 ± 7.45 mg/dl compared to 28.52 ± 3.02 mg/dl in wild type (WT) mice), serum creatinine (0.53 ± 0.08 mg/dl compared to 0.30 ± 0.04 mg/dl in WT mice) and elevated level of serum Pi (9.4 ± 1.21 mg/dl compared to 6.1 ± 0.38 mg/dl in WT mice). We have found by WB that there is an increase in intestinal NaPi‐2b protein abundance in the ileum of the Alport mice. Everted Sac studies revealed increased NaPi transport in the Alport Ileum compared to the WT littermate controls. Our study indicates an important role for increased intestinal Pi absorption and NaPi‐2b protein in hyperphosphatemia in CKD.
Neural FFA3 Negatively Regulates Cholinergic Secretory Function in Rat Proximal Colon
Authors: Kaji Izumi , Akiba Yasutada , Narimatsu Kazuyuki , Kaunitz Jonathan
Published: June 2021

The lumen of the proximal colon is continuously exposed to high concentrations of short‐chain fatty acids (SCFAs). Although propionate evokes transient anion secretion via neural and non‐neural cholinergic pathways in the colon, the involvement of FFA3 (GPR41) and its absorptive pathway have not been clarified. We investigated the expression of FFA3 and its contribution towards propionate‐ and carbachol (CCh)‐induced secretory responses in rat proximal colon. FFA3 immunolocalized to the enteroendocrine cells and to the enteric neural plexuses. Most of the FFA3‐immunoreactive nerve fibers were cholinergic, based on staining with ChAT, a vesicular ACh transporter, and the high affinity choline transporter CHT1. In Ussing chambered proximal colon, luminal propionate induced anion secretion in fasted, but not fed conditions. TTX or atropine reduced the response to propionate by 60% or 94%, respectively. In the presence of indomethacin, the non‐substrate sodium‐dependent monocarboxylate transporter (SMCT)1 inhibitor ibuprofen or fenoprofen abolished the response to propionate, without inhibition on CCh‐evoked secretion. CCh‐induced secretion was decreased by TTX or serosal propionate by 40%. Serosal application of the FFA3 agonist decreased the response to propionate and CCh, indicating that FFA3 negatively regulates cholinergic neural reflexes. These results suggest that luminal propionate‐induced anion secretion is dependent on SMCT1‐mediated SCFA absorption, which in turn varies cholinergic nerve activity.

Supported by VA Merit Review, NIH R01 DK54221
Klotho/fgf23‐Independent and ERα Mediated Downregulation of NaPi‐IIa and NaPi‐IIc by Estrogen in the Mouse Kidney Proximal Tubule
Authors: Webster Rose , Sheriff Sulaiman , Faroqui Rashma , Siddiqui Faraaz , Hawse John R. , Amlal Hassane
Published: June 2021

In these studies, we examined the effect of estrogen on renal inorganic phosphate (Pi) transport and the roles of its receptor isoforms (ERα and ERβ) and Klotho/FGF23 pathway in this effect in mice. Mice with null mutations (KO) of ERα or ERβ or Klotho and their wild‐type (WT) were placed in metabolic cages and injected daily with 17β‐estradiol or vehicle for 3 days. Food intake, urine volume and Pi excretion were measured daily. The results indicate that estrogen‐treated WT mice exhibited a significant increase in renal Pi wasting despite a reduction in food intake. Molecular studies showed a significant downregulation of NaPi‐IIa and NaPi‐IIc proteins with no change in their mRNA expression levels in the kidney. Estrogen‐induced reduction in food intake, phosphaturia and downregulation of NaPi‐IIa and NaPi‐IIc proteins were observed in ERβ KO but not in ERα KO mice. Further, Klotho KO mice exhibited a significant downregulation of NaPi‐IIa and hypophosphatemia in response to estrogen. In a cell line stably co‐expressing both ERα and ERβ, 24h of estrogen downregulated NaPi‐IIa protein, when cells are transiently transfected with a plasmid containing ORF‐3'UTR but not 5'UTR‐ORF of the mouse NaPi‐IIa transcript. In conclusion, estrogen causes phosphaturia in mice through the downregulation NaPi‐IIa and NaPi‐IIc protein abundance in the kidney. This effect is independent of Klotho/FGF23 pathway and is mediated through the activation of ERα. 3'UTR region of mouse NaPi‐IIa mRNA likely plays an important role in the inhibition of its translation by estrogen.
Renal Caffeine Effects are Independent of NHE3 Abundance, Trafficking or Phosphorylation
Authors: Rieg Timo , Soleimani Manoocher , Dominguez Rieg Jessica , Busslinger Meinrad , Fenton Robert
Published: June 2021

We have previously shown that the diuretic and natriuretic effect of caffeine is mediated by blockade of adenosine A1 receptors and both effects are unaffected in the absence of renal tubular NHE3. However, compensatory mechanisms in tubulus‐specific NHE3 knockout mice may mask caffeine effects on NHE3 abundance, trafficking or phosphorylation. Wild‐type mice were randomized to acute application of caffeine (45 mg/kg, n=6) or water (vehicle, n=6) via oral gavage (1% of bw) and kidneys harvested after 60 min. Renal NHE3 protein abundance (vehicle: 100±8, caffeine: 101±13 %), phosphorylation at serine 552 (vehicle: 100±14, caffeine: 86±10 %) or serine 605 (vehicle: 100±8, caffeine: 96±9 %) were comparable between groups. Membrane abundance analyzed via semi‐quantitative confocal laser scanning microscopy showed no significant difference between groups. We hypothesized that the Na+/HCO3‐ cotransporter NBC1 (NBCe1) may be responsible for mediating the caffeine effects. Mice were treated with vehicle (0.85% NaCl, n=12) or DIDS (NBC inhibitor, 40 mg/kg i.p., 2 µl/g bw, n=10) 30 min before application of caffeine (45 mg/kg, 3% of bw by oral gavage), and a 3 hour urine collection performed. DIDS+caffeine resulted in a significantly lower urinary flow rate vs vehicle+caffeine (191±31 vs 296±16 nl/min/g, P<0.05) which was not different from vehicle alone (203±19 nl/min/g, n=12). Our data show that NHE3 is not required for caffeine‐induced renal effects but point towards a role of NBC1 in mediating caffeine‐induced diuresis.
Activation of Brain Angiotensin Type‐2 Receptors Reverses DOCA‐salt Hypertension in Mice
Authors: de Kloet Annette , Wang Lei , Sumners Colin , Krause Eric
Published: June 2021

These studies assess the impact of activating brain angiotensin type‐2 receptors (AT2R) on blood pressure in a mouse model of hypertension. Experiments combine the use of a transgenic AT2R green fluorescent protein reporter mouse with in situ hybridization (ISH) to reveal that AT2R‐containing cells are localized to brain regions that regulate cardiovascular function. In particular, the nucleus of the solitary tract (NTS) densely expresses AT2R, primarily in glutamic acid decarboxylase‐1 (Gad1)‐containing (i.e., GABAergic) neurons. Based on this localization and the known hypertensive actions of GABA in the NTS, we hypothesized that brain AT2R activation would reverse hypertension in mice via the inhibition of NTS GABA signaling. Mice were initially rendered hypertensive via administration of deoxycorticosterone acetate and consumption of isotonic saline (DOCA‐salt) or served as normotensive controls. Subsequently, the impact of the chronic administration of the selective AT2R agonist, compound 21 (C21; 7.5 ng/h into the lateral cerebral ventricle [icv]) on blood pressure and gene expression within the NTS were assessed. Chronic icv C21 reduced baseline blood pressure relative to control mice and reversed DOCA‐salt hypertension. Gene expression studies revealed that DOCA‐salt hypertension was associated with elevated Gad1 and AT2R mRNAs within the NTS. The antihypertensive actions of C21 were associated with decreased Gad1 mRNA in the NTS, suggestive of reduced GABAergic signaling. Collectively, the present results indicate that AT2R activation using C21 reverses DOCA‐salt hypertension and that this is accompanied by alterations in NTS gene expression that are consistent with NTS GABAergic signaling playing a role in these effects.
Lipoxin A4 Inhibits Histamine‐stimulated Increase in [Ca2+] and Mucin Secretion in Cultured Rat and Human Goblet Cells
Authors: Hodges Robin , Li Dayu , Bair Jeffrey , Shatos Marie , Chiang Nan , Serhan Charles , Dartt Darlene
Published: June 2021

In allergic conjunctivitis histamine levels are elevated and goblet cell mucin secretion stimulated. The purpose of this study was to determine if the effects of histamine can be blocked by the pro‐resolution compound lipoxin A4 (LXA4) in cultured conjunctival goblet cells (GCs), similar to pro‐resolution effects of resolvin D1 (RvD1). First passage GCs from rat and human were used. Intracellular [Ca2+] ([Ca2+]i) was measured using fura2. LXA4 (10‐11‐10‐8M) was added alone or 0.5h prior to histamine (10‐5M). In rat GCs, LXA4 alone increased [Ca2+]i a maximum of 116.4±21.5 nM above basal which was significantly inhibited by the ALX receptor inhibitor BOC‐2. In human GCs, LXA4 increased [Ca2+]i a maximum of 762.0±259.4nM above basal which was inhibited by BOC‐2. LXA4 inhibited histamine‐stimulated increase in [Ca2+]i by 63.7±10.2% and 71.0±7.7% in rat and human CGs, respectively. In rat GCs, stimulation with the specific H1 receptor agonist histamine dimaleate stimulated [Ca2+]i by 751.7±216.1 nM. This was inhibited by LXA4 by 70.5±8.9%. Preincubation with an inhibitor to b‐adrenergic receptor kinase 1 (βARK1), but not inhibition of protein kinase C by Ro31754, reversed LAX4 inhibition of histamine dimaleate response. We conclude that similarly to RvD1, LXA4 functions as a pro‐resolution mediator as it inhibits histamine‐stimulated responses by counter‐regulation of the H1 histamine receptor through βARK1. LXA4 could play a role in the return of GCs to homeostasis in allergic conjunctivitis.
Short‐ and Long‐term Regulation of Intestinal Na+/H+ exchange by TLR4 in intestinal epithelial cells
Authors: Cabral Jose , Grácio Daniela , Soares‐da‐Silva Patricio , Magro Fernando
Published: June 2021

The ability of recognizing microbiological infections is largely dependent on toll‐like receptors (TLR). TLR4 is expressed in the cell membrane and is activated by bacterial lipopolysaccharide, one of components of the outer membrane of Gram‐negative bacteria.

Aims

To evaluate the effect of TLR4 activation by Monophosphoryl Lipid A (MPLA) on Na+/H+ exchanger (NHE) activity in T84 cells as well the signal transduction set in motion.

Material and methods

NHE activity and kinetics were measured using fluorimetric assays and protein expression was determined by western blot analysis.

Results

Short‐ and long‐term MPLA (0.01 to 50 µg/ml) exposure induced an NHE1 concentration dependent inhibition (st ‐4.9±5 to ‐25.2±5 %; lt ‐3.9± to ‐31.9±4 %) without any effect over NHE membrane protein expression. The cell signal triggered AC3, PKA, PLC and PKC signal molecules. Long‐term exposure induced changes in NHE activity but affecting different NHE isoforms depending on MPLA concentration, lower MPLA induced NHE3 inhibition and higher MPLA concentrations induced NHE1 inhibition. In both cases AC3, PKA, PLC and PKC are involved. That is, the final effector of NHE1 was PCK and for NHE3 was PKA. MPLA induced an impaired recover of pHi and consequent acidification of intracellular media.

Conclusions

Activation of TLR4 exerts marked inhibition of both NHE1 and NHE3 activity in intestinal epithelial cells. Transduction mechanisms set into motion during short‐ and long‐term TLR4‐mediated inhibition of NHE1 activity involves AC and PKA. However, short‐ and long‐term TLR4 activation might use different signalling pathways.
Expression of Cdk5r1 and not Cdk5r induces primary β‐cell proliferation
Authors: Bitner Ben , Draney Carrie , Hobson Amanda , Tessem Jeffery
Published: June 2021

Decreased β‐cell mass is a hallmark of type 1 and type 2 diabetes. The use of islet transplantation as a diabetes therapy is hampered by the relative paucity of transplant ready islets. Greater understanding of the proliferative pathways controlling islet proliferation may be harnessed to increase functional beta‐cell mass through transplantation or by enhanced growth of endogenous beta‐cells. We have shown that the β‐cell transcription factor Nkx6.1 induces β‐cell proliferation by upregulating the orphan nuclear hormone receptors Nr4a1 and Nr4a3. Using expression analysis to elucidate the Nkx6.1 independent mechanism by which Nr4a1 and Nr4a3 induce beta‐cell proliferation, we demonstrated that cyclin‐dependent kinase 5 regulatory subunit 1 (Cdk5r1) is upregulated by Nr4a1 and Nr4a3 and not Nkx6.1. Adenovirus mediated overexpression of Cdk5r1 is sufficient to induce proliferation in primary rat islets. The observed proliferation is primarily in beta‐cells. Glucose stimulated insulin secretion is maintained with Cdk5r1 overexpression. The Cdk5 inhibitor roscovitine blocks islet proliferation, suggesting that Nr4a mediated β‐cell proliferation is a kinase dependent event. Overexpression of Cdk5r1 results in pRb phosphorylation, which is inhibited by roscovitine treatment. These data demonstrate that activation of the cdk5 complex is sufficient to induce β‐cell proliferation while maintaining glucose stimulated insulin secretion.
Skeletal Muscle β‐F1‐ATPase Translation is Inhibited by Hyperlipidemia‐Induced miR‐127‐5p Expression in Human Obesity
Authors: Tran Lee , Campbell Latoya , Coletta Dawn , Mandarino Lawrence , Katsanos Christos
Published: June 2021

We recently discovered persistent downregulation of the H+‐ATP synthase catalytic subunit (β‐F1‐ATPase) in skeletal muscle of obese individuals, which was linked to comorbid hyperlipidemia. β‐F1‐ATPase expression is primarily regulated post‐transcription, i.e. miRNAs, but the precise mechanism impeding expression are unclear. We tested the hypothesis that hyperlipidemia reduces β‐F1‐ATPase translational efficiency and is mediated by miR‐127‐5p, a miRNA known to control β‐F1‐ATPase translation. Biopsies isolated from the vastus lateralis of lean (BMI蠄25; n=7) and obese (BMI蠅30; n=6) subjects were evaluated for mRNA/miRNA expression via qRT‐PCR, protein by Western blot, and translation efficiency was calculated as the mRNA/protein ratio. Our results confirmed β‐F1‐ATPase translation was lower in obese individuals (P<0.01; 0.25±0.05%) compared to healthy weight controls (0.59±0.09%), and correlated with miR‐127‐5p expression (P<0.01; r=‐0.67). Bisulfite sequencing analysis of the miR‐127‐5p promoter revealed two hypomethylated sites in obese individuals, which may sustain an upregulation. Moreover, we found miR‐127‐5p present in muscle‐derived blood exosomes suggesting putative involvement in intracellular cross‐talk. In primary myotubes from a lean volunteer, prolonged fatty acid exposure increased miR‐127‐5p expression.

Free Fatty Acid (mEq/L)
Unsaturated/ Saturated
mRNA (2‐DDCt)
Protein (AU)
Translation (%)

CTRL
0.347
‐
1.70±0.113
0.216±0.062
0.130±0.040

Lo Fat
0.185
0.55
1.60±0.094
0.202±0.038
0.123±0.008

Hi Fat
0.251
2.29
1.35±0.133*
0.095±0.006*
0.073±0.010

Hi Fat
0.269
0.54
1.66±0.145
0.093±0.003*
0.050±0.004*

*P<0.05; One‐way ANOVA, Tukey9s posttest

Inhibiting miR‐127‐5p with locked nucleic acids dose‐dependently improved β‐F1‐ATPase translation.

Inhibitor (nM)
mRNA (2‐DDCt)
Protein (AU)
Translation (%)

0
1.84±0.314
0.22±0.062
0.647±0.120

10
1.93±0.387
1.24±0.135*
0.500±0.148

50
2.00±0.498
1.24±0.242**
1.483±0.233*

100
1.76±0.207
1.54±0.086*
0.892±0.096

*P<0.05; **P<0.01; ***P<0.001; 1‐way ANOVA, Tukey9s posttest

Our results provide evidence implicating hyperlipidemia‐induced miR‐127‐5p expression in suppressing β‐F1‐ATPase translation in human obesity.

Funding from NIH/NIDDK grant DK094062 to CSK
Taming the Promoter: Regulation of Tissue Inhibitor of Matrix Metalloprotease 4 in Heart Failure
Authors: Chaturvedi Pankaj , Tyagi Suresh
Published: June 2021

The remarkable property of tissue inhibitor of matrix metalloprotease 4 (TIMP4) to inhibit endogenous MMP9 potentiates its use in cardiac remodeling prior to heart failure. This concept stems from our previous discovery that TIMP4 goes down in heart failure and administration of TIMP4 intraperitonialy is cardio protective. This important finding instigated us to explore the differential regulation of TIMP4 during heart failure. We hypothesize that there are epigenetic changes in the promoter of TIMP4 pertaining to DNA methylation of CpG islands, acetylation of histone proteins and binding of microRNAs (mir122a) that lead to epigenetic silencing of TIMP4 and enhanced expression of MMP9 which consequently lead to heart failure. To test the hypothesis we created heart failure by aorta‐vena cava fistula (AVF) in 1) C57BL/6 mice 2) TIMP4‐Tg mice and examined the promoter methylation (methylation specific PCR, HRM and methylation sensitive restriction enzyme), histone modification (ChIP assay) and microRNAs (mir122a that binds to TIMP4). The cardiac function was assessed by echocardiography after 6 weeks. The data suggested that the promoter region of TIMP4 was methylated in the AVF failing heart, suggesting epigenetic silencing of TIMP4. To determine the contribution of epigenetic silencing by turning off the transcription machinery, we observed methylation of histone 3 at the lysine 9. We discovered that the CpG islands get methylated during heart failure with decreased acetylation of histone 3 at lysine 9 and 27. This is a novel finding suggesting that TIMP4 is epigenetically silenced in failing hearts and can be translated to regulate its expression prior to heart failure.
Aliskiren Modifies Canonical Wnt/β‐Catenin Signaling Pathway Associated to (Pro)renin Receptor on Heart and Kidney in 5/6 Nephrectomy‐Induced Hypertension in Rats
Authors: Lopez‐Sanchez Pedro , Avila‐Ramirez Massiel , Ramirez‐Montero Claudia , Anguiano‐Robledo Liliana
Published: June 2021

The Wnt/frizzled receptor signaling pathways are important in morphogenesis and development of essential organs. (Pro)renin receptor (PRR) has been characterized as a multifunctional protein associated with vacuolar H+‐ATPase (v‐H+‐ATPase) (independently of renin) and is required for canonical Wnt/β‐catenin signaling activation. Aliskiren, a direct renin inhibitor, has been used to treat cardiovascular disorders. The aim of this work was to study Aliskiren effects upon PRR and canonical Wnt/β‐catenin pathway in cardiac and renal tissue during 5/6 nephrectomy (5/6 Nx)‐induced hypertension. Male Wistar rats underwent 5/6 Nx or sham operation (S), and were treated with Aliskiren, 10 mg/kg/day/7 days. Control groups received vehicle. 24‐hour urine was collected and blood pressure was measured at the end of experiment. PRR, β‐catenin and dishevelled (Dvl‐1) expression was determined by immunoblot. Our results showed that, compared to sham animals, 5/6 Nx rats treated with Aliskiren had lowered blood pressure and improved seric creatinine, proteinuria and creatinine clearance. We found in nephrectomized rats that PRR expression increased in heart and kidney seven days after surgery, and aliskiren reduced PRR, β‐catenin and Dvl‐1 expression in both tissues. These results confirm the antihypertensive and renoprotective effects of aliskiren and interestingly, showed that aliskiren modifies PRR expression as well as canonical Wnt/β‐catenin signaling expression. Supported by SIP20140636 grant
Chronic Alcohol Consumption and its Effects on the Cardiac Apoptosis
Authors: Etoundi Clementine , Haddad George , Mark Burke
Published: June 2021

Alcohol is a product that has been used by virtually all cultures for rituals, worship, antibacterial and analgesic properties. Clinically, excessive alcohol use increases the risk of liver and heart disease, and other facets of morbidity and mortality. Alternatively, low alcohol has been shown to possess protectant cardiovascular properties while high alcohol has been linked to heart pathology. This experiment focused on potential impact of apoptosis interceding with cardiac effects of chronic high alcohol versus low alcohol exposure. Fraternal rats were pair fed with a liquid diet (Liber‐Dicarli) supplemented with either none, low or high alcohol (5mM or 100mM EtOH) for 5 months. Anesthetized with 5% halothane and excised for retrograde perfusion with PBS, after which hearts were fixed with PFA, and mounted on the cryostat. 30 micron sections of hearts tissue were put in wells with antifreeze, others placed on slides and stained using the cresyl violet procedure. Immunohistochemistry with caspase‐3/7 antibody was performed on free floating sectioned heart tissue from the high and low alcohol heart and control groups to visualize the effects on apoptosis. Chronic high alcohol caused a thinning of left ventricular wall and the enlargement of the ventricle lumen compared to chronic low alcohol and control groups. The caspase‐3/7 immunoreactivity was evident in papillary muscle and endocardium but less evident in the myocardium and epicardial layers. Chronic high alcohol consumption activated adverse cardiac effects initiated by endocardial apoptosis while chronic low alcohol enhanced cellular capacity for survival.
Cytochrome P450 ω‐hydroxylase and COX‐2 Inhibition Reduces Stretch‐Induced Renal Fibrosis in HK‐2 Cells
Authors: Avedissian Hari , Farhat Kalil , Alexander Larry
Published: June 2021

Fibronectin matrix deposition is a hallmark of renal fibrosis, which ultimately leads to end‐stage renal failure. We have previously shown that the expression of fibronectin is increased by arachidonic acid in human renal proximal tubular epithelial (HK‐2) cells. Here we found that cyclic mechanical stretch up‐regulated protein levels of fibronectin by using Western blotting analysis. Using pharmacological inhibitors of phospholipase A2 (PLA2) or arachidonic acid metabolic pathways revealed that cytosolic phospholipase A2 (cPLA2), cytochrome P450 (CYP) ω‐hydroxylase, and cyclooxygenase‐2 (COX‐2) isoenzymes are involved in mechanical stretch‐induced up‐regulation of fibronectin. Indeed, mechanical stretch significantly increased 20‐hydroxyeicosatetraenoic acid (20‐HETE) and prostaglandin E2 (PGE2) production, metabolites of CYP ω‐hydroxylase and COX‐2, respectively, compared with control. Likewise, exogenous 20‐HETE or PGE2 administration also elicited an increase in fibronectin expression. In line with this, the selective inhibitor of 20‐HETE synthesis HET0016 and COX‐2 inhibitor NS398 prevented fibronectin synthesis in response to mechanical stretch. In western blotting analysis, mechanical stretch increased the activity of ERK1/2, p38 MAPK, and JNK and PD 98059 or SB 203580 blocked the effect of mechanical stretch on fibronectin expression. These data provide the first evidence, in HK‐2 cells, of production of fibronectin by cyclic mechanical stretch. Moreover, our results provide evidence that blockade of CYP ω‐hydroxylase and COX‐2 may provide significant anti‐fibrotic therapy for treatment of obstructive nephropathy.
Sepiapterin Attenuates Increased Mitochondrial Fission in Human Cardiomyocytes Exposed to High Glucose
Authors: Williams Anna , Olson Jessica , Canfield Scott , Bai Xiaowen , Bosnjak Zeljko
Published: June 2021

Anesthetic cardioprotection by volatile anesthetics is shown to reduce the infarct size after a myocardial ischemia/reperfusion injury. This protective effect is abolished under diabetic conditions, but the causal mechanisms have not been completely elucidated. Previously, we have demonstrated that high glucose increased reactive oxygen species (ROS) generation and mitochondrial fission. Sepiapterin, a BH4 analog, could decrease ROS by increasing nitric oxide synthase. In this study, we examined the relationship between mitochondrial fission and NO signaling pathways. We hypothesized that modulation of BH4/NO signaling with sepiapterin can restore the attenuated anesthetic cardioprotection conferred by high glucose through decreasing mitochondrial fission.

Human induced pluripotent stem cell derived cardiomyocytes (CMs) were exposed to 5 or 25 mM glucose for 3 days with or without sepiapterin. CMs were exposed to isoflurane for 30 minutes followed by 6 hours of H2O2 exposure. Cell viability was measured using a lactate dehydrogenase assay or propidium iodide staining. Mitochondria were loaded with Mitotracker Green and imaged using confocal microscopy. The images were analyzed using the ImageJ software to assess the form factor and aspect ratio.

CMs in 5 and 11 mM glucose were protected against oxidative stress as demonstrated by increased cell viability and decreased mitochondrial fission in isoflurane‐treated CMs. However, CMs exposed to 25 mM glucose had an attenuation of cardioprotection and increased mitochondrial fission. Addition of sepiapterin reduced mitochondrial fission and increased cell viability in CMs cultured in 25 mM glucose.
Decreased nitric oxide (NO) Availability Contributes to the Impaired Mitochondrial Biogenesis and Oxidative Stress in Fetal Lambs with Pulmonary Hypertension (PPHN)
Authors: Afolayan Adeleye
Published: June 2021

We previously showed increased oxidative stress and ATP depletion in PPHN. We hypothesize that decreased NO levels and increased mitochondrial oxidative stress impairs mitochondrial function in PPHN.

Methods‐ PAECs were isolated from control fetal lambs and lambs with PPHN induced by prenatal constriction of the ductus arteriosus. We knockdown eNOS in control PAEC using siRNA, and treated PPHN PAEC and eNOS depleted cells with 200µM detaNONOate (deta), an NO donor, for 18h. We quantified mtDNA copy number, electron transport chain subunits complexes I‐V (CI‐V) and ATP levels at baseline and following deta treatment using rT‐PCR, WB and bioluminescence assay. Levels of PGC1α, Sirti1, and p‐AMPKα were also assessed by WB. Effects of NO on basal O2 consumption rate (OCR) and mitochondrial superoxide (mtO2̅) in PPHN were assessed using dihydroethidium and seahorse XF24 assay. We also assess changes in SOD2 expression and specific activity following deta treatment in PPHN PAEC using WB, Cayman activity assay and immunoprecipitation.

Results‐MtDNA copy number is decreased in PPHN. Levels of PGC1α, Sirti1 and p‐AMPKα were also decreased. This is accompanied by decreased CI‐V and ATP levels. eNOS knockdown decreased CI‐V levels in control PAEC. Deta increased mtDNA copy number, CI‐V and ATP levels in PPHN and in eNOS depleted cells. Basal OCR is decreased albeit, ATP‐linked O2 consumption is increased in deta treated PPHN‐PAEC. Increased bioenergetics is accompanied lower mtO2‐ levels and increased SOD2 expression and activity.

Conclussion‐Alteration in mitochondrial metabolism is a key feature of PPHN, and has important therapeutic implications. In PPHN, NO restores mitochondrial biogenesis and decreases mtO2̅ levels.
Elevated Apoptosis in Ovine Intrauterine Growth Restriction (IUGR) is Associated with Increased Caspase 3 and 9 and Decreased Telomerase Activity
Authors: Mika Aleksander , Reynolds Paul , Arroyo Juan
Published: June 2021

Intrauterine growth restriction (IUGR) has been documented to increase placental apoptosis at term. Previous reports from our lab showed apoptosis as an early event in an ovine model of IUGR. Our objective was to determine the placental expression of apoptotic proteins and telomerase activity at early (55 days of gestation; DGA), mid (95 dGA) and near term gestation (130 dGA) in a hyperthermia (HT) induced model of IUGR. Pregnant sheep were placed in hyperthermic (HT) conditions and placental tissues were collected at 55, 95 and 130 dGA. Compared to controls we found: 1) increased telomerase activity at day 55 dGA (1.3‐fold; p <0.007) with decreased activity at 130 dGA (1.3‐fold;p <0.02); 2) increased cleaved caspase 9 only at 95 dGA (1.6‐fold; p <0.008); 3) no significant differences for BAX protein across gestation; 4) increased caspase 3 at 95 and 130 dGA; and 5) decreased Bcl2 protein at 55 dGA (1.6‐fold; p < 0.02) with decreased expression at 95 dGA (1.4‐fold; p< 0.02). We conclude that elevated placental apoptosis near term coincides with decreased telomerase activity, increased caspase 3, and inhibition of active Bcl2. These data suggest that several mitochondrial pro‐apoptotic genes contribute to the induction of placental apoptosis during IUGR.
Differential Effects of Buffer pH, CaCl2, and Superoxide Dismutase on Ca2+‐Induced H2O2 Release from Mitochondrial Complexes I and III
Authors: Lubbe Ryan , Lindsay Daniel , Aldakkak Mohammed , Camara Amadou , Stowe David
Published: June 2021

Mitochondrial dysfunction underlies many aspects of cardiovascular disease, but the role mitochondria play in cardiac ischemia/reperfusion (IR) injury is uncertain. During IR injury, electron transport chain complexes I and III are considered primary sources of reactive oxygen species (ROS). Increased extra‐matrix Ca2+, decreased extra‐matrix pH, and substrate utilization change contribute to enhancing ROS emission during ischemia. Studies in isolated mitochondria show ROS emission is modulated by buffer pH or extra‐matrix [CaCl2] with complex I substrate pyruvate (P) or complex II substrate succinate (S). We tested the combined effects of these factors on mitochondrial H2O2 release. Guinea pig heart mitochondria were suspended in buffer of varied pH (7.15, 6.9, or 6.5), ± exogenous superoxide dismutase (SOD), and ± 150 µM CaCl2 (+40 µM EGTA). Mitochondria were energized with P and rotenone (R, complex I blocker) or S and antimycin A (AA, complex III blocker). H2O2 release rate was compared for P+R and S+AA. For P+R, there were no significant differences in H2O2 release at all pHs with added SOD and no added CaCl2. However, H2O2 release increased at pH 7.15 and 6.9 ± SOD after adding CaCl2. In contrast, with S+AA there were no significant differences in H2O2 release at all pHs when exogenous SOD was added to the experimental buffer and incubated ± added CaCl2; however, at 150 µM CaCl2 H2O2 release increased at each pH. These results suggest a higher extra‐matrix pH (7.15, 6.9), coupled with a large increase in extra‐matrix [CaCl2], may play a role in enhancing SOD activity to dismutate O2•‐ generated at complexes I and III to H2O2. In contrast, at an acidic pH (6.5) a rise in [Ca2+] may have little effect on SOD activity.
Early neonatal but not late neonatal sevoflurane inhalation induced special memory impairment revealed by behavioral investigation of mouse
Authors: Hata Kanato , Maekawa Masao , Takasusuki Toshifumi , Yamaguchi Shigeki , Hori Yuuichi
Published: June 2021

Sevoflurane is a volatile anesthetic that was developed relatively recently, and is now widely used in clinical anesthesia for infants. Sevoflurane has proposed effects on GABAa receptor‐mediated extrasynaptic tonic inhibition. Recent reports have shown that exposure to sevoflurane at an early age lead to long‐term cognitive dysfunction using animal models. However, the mechanisms underlying the sevoflurane induced cognitive dysfunction have not been fully understood. Aiming to find the mechanisms we observed behavioral changes of adult mice after the exposure to sevofrulane at different neonatal stage to define the period during which the sevoflurane exposure induces the long‐term cognitive dysfunction. Neonatal mice (3‐5 days, 12days, 20 days after birth) were exposed to 2% sevoflurane in O2 for 4hrs. These mice were trained for 4 days (6 trials/day) from 49 days after birth for water maze test using a 1.2m diameter pool with a 10cm diameter platform. The escape latencies and the percent time of swim in the platform quadrant in the probe test for 60 sec was measured as indices of spatial memory formation. Mice exposed to sevoflurane at P3‐5 showed impaired water maze performance as compared with control mice. On the other hand, mice exposed to sevoflurane at P12 or P20 showed water maze performance comparable to the control mice. Our results suggest that during early neonatal period (around 3‐5 days after birth) the brain mechanisms relating spatial memory are sensitive to the sevoflurane exposure, but during later neonatal period (12 or 20 days after birth) those are not sensitive to the exposure.
Expression of the D2 and D5 dopaminergic receptors related to non‐caloric commercial sweetener intake
Authors: Contreras Irazú , Ochoa‐Arriaga Fany , Sotuyo‐González Alejandra , Estrada José
Published: June 2021

Human beings have a preference for sweet flavors, which are mostly achieved by the use of sugar in food. Addition of non‐caloric sweeteners to food and beverages is presently a common practice, seeking to obtain the flavor without the caloric content and therefore, reducing the risk of developing non‐communicable chronic diseases, such as obesity and type 2 diabetes. The use of sweeteners to promote a healthy diet and weight control is broadly accepted, but there is currently evidence that contradicts such ideas, demonstrating that the use of some of these compounds may alter the hunger‐satiety system. Thus, it is necessary to regulate the quantity and duration of sweetener intake to evaluate the effect they might have on molecules that regulate central and peripheral signals of the hunger‐satiety system. Expression of dopaminergic receptors D2 and D5, which have a role in the neuroendocrine regulation of food and water intake, was evaluated in 14 week‐old BALB/c mice that consumed either sucrose, stevia or sucralose in their daily water for 6 weeks, by performing a western blot analysis of proteins from their brains. Our preliminary results show a higher expression of D2 in the brains of mice in the sucralose group, whereas D5 presented higher expression in the sucrose and sucralose samples. Our data suggest that chronic intake of commercial non‐caloric sweeteners may alter the expression of dopaminergic receptors and modify the signals that control the normal functions of the hunger‐satiety system.
In vivo MEMRI Reveals Decreased Activation of the Hippocampal and Amygdaloid Regions in a Rodent Model of RAS‐Dependent Hypertension
Authors: Zubcevic Jasenka , Perez Pablo , Ahmari Niousha , Marulanda Carvajal Jessica , Raizada Mohan , Febo Marcelo
Published: June 2021

Brain RAS affects pathogenesis of neuronal disease by contributing to neuroinflammation in animal models including hypertension. Thus, we hypothesized that the overactive brain RAS will directly contribute to decreased neuronal activity in cognitive brain areas. METHODS: Manganese‐enhanced magnetic resonance imaging (MEMRI) yielded in vivo mapping of spontaneous neuronal activity in the amygdala and hippocampus in a rodent model of overactive brain RAS and neuroinflammation, the spontaneously hypertensive rat (SHR) compared to the Wistar Kyoto (WKY). Rats were treated with manganese chloride (MnCl2 30 mM, i.p.; 16‐20 hrs) and T1‐weighted images were obtained (4.7T). Coronal slice scans were automatically segmented (FMRIB FSL and Ekam Solutions) and analyzed for differences in normalized signal intensity. RESULTS: We observed lower signal intensity in basal/central amygdala (53%), lateral (73%) and medial amygdala (50%), CA2 (46%) and CA3 hippocampal nuclei (30%), central gray (49%), and habenula (15%), in the SHR vs WKY rats.

CONCLUSION

To our knowledge, this is the first MEMRI assessment of neuronal activity suggesting abnormal hippocampal and amygdaloid neurocognitive functioning in the SHR. This supports the role of hypertension in cognitive decline and advocates for a therapeutic approach for neuronal disease by regulating the brain RAS.NIH HL33610.