Mid-Point of the Active Phase Is Better to Achieve the Natriuretic Effect of Acute Salt Load in Mice.

Excess sodium intake and insufficient potassium intake are a prominent global issue because of their influence on high blood pressure. Supplementation of potassium induces kaliuresis and natriuresis, which partially explains its antihypertensive effect. Balancing of minerals takes place in the kidney and is controlled by the circadian clock; in fact, various renal functions exhibit circadian rhythms. In our previous research, higher intake of potassium at lunch time was negatively associated with blood pressure, suggesting the importance of timing for sodium and potassium intake. However, the effects of intake timing on urinary excretion remain unclear. In this study, we investigated the effect of 24 h urinary sodium and potassium excretion after acute sodium and potassium load with different timings in mice. Compared to other timings, the middle of the active phase resulted in higher urinary sodium and potassium excretion. In Clock mutant mice, in which the circadian clock is genetically disrupted, urinary excretion differences from intake timings were not observed. Restricted feeding during the inactive phase reversed the excretion timing difference, suggesting that a feeding-induced signal may cause this timing difference. Our results indicate that salt intake timing is important for urinary sodium and potassium excretion and provide new perspectives regarding hypertension prevention.

Renomedullary exosomes produce antihypertensive effects in reversible two-kidney one-clip renovascular hypertensive mice.

The rapid fall in blood pressure following unclipping of the stenotic renal artery in the Goldblatt two-kidney one-clip (2K1C) model of renovascular hypertension is proposed to be due to release of renomedullary vasodepressor lipids, but the mechanism has remained unclear. In this study, we hypothesized that the hypotensive response to unclipping is mediated by exosomes released from the renal medulla. In male C57BL6/J mice made hypertensive by the 2K1C surgery, unclipping of the renal artery after 10 days decreased mean arterial pressure (MAP) by 23 mmHg one hr after unclipping. This effect was accompanied by a 556% increase in the concentration of exosomes in plasma as observed by nanoparticle tracking analysis. Immunohistochemical analysis of exosome markers, CD63 and AnnexinII, showed increased staining in interstitial cells of the inner medulla of stenotic but not contralateral control kidney of clipped 2K1C mice. Treatment with rapamycin, an inducer of exosome release, blunted the hypertensive response to clipping, whereas GW-4869, an exosome biosynthesis inhibitor, prevented both the clipping-induced increase in inner medullary exosome marker staining and the unclipping-induced fall in MAP. Plasma exosomes isolated from unclipped 2K1C mice showed elevated neutral lipid content compared to sham mouse exosomes by flow cytometric analysis after Nile red staining. Exosomes from 2K1C but not sham control mice exerted potent MAP-lowering and diuretic-natriuretic effects in both 2K1C and angiotensin II-infused hypertensive mice. These results are consistent with increased renomedullary synthesis and release of exosomes with elevated antihypertensive neutral lipids in response to increased renal perfusion pressure.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

The downstream RAF-1 signaling of fibroblast growth factor-23 participates in the osteogenetic effect caused by C-type natriuretic peptide in vitro.

PURPOSE: Several studies have demonstrated that C-type natriuretic peptide (CNP) stimulates osteoblastic proliferation seemly via antagonizing the expression of fibroblast growth factor (FGF)-23 in vitro. The main aim of the present study is to probe whether the post-receptor pathways of FGF-23 participate in osteogenesis caused by CNP. METHODS: Osteoblasts were cultured in the absence or presence of CNP: 0, 10, and 100 â€‹pmol/L, for 24 â€‹h, 48 â€‹h and 72 â€‹h, respectively. RESULTS: The findings of the present study indicated that osteoblastic proliferation was directly promoted by exogenous CNP in a dose-dependent manner; osteoblastic FGF-23 was significantly down-regulated by CNP at 24 â€‹h post-treatment; RAF-1, extracellular signal-regulated kinases (ERK), and P38 were substantially suppressed by CNP in a dose- and time-dependent manner; and signal transducer and activator of transcription (STAT)-1 was not changed on the premise of the down-regulated FGF-23 in osteoblasts treated with CNP. CONCLUSION: CNP may promote osteogenesis via inhibiting ERK and P38, rather than STAT-1, in the downstream of FGF-23/RAF-1 pathway.

Treatment of cattle oocytes with C-type natriuretic peptide before in vitro maturation enhances oocyte mitochondrial function.

The present study was conducted to evaluate the effects in vitro on oocyte mitochondrial function of C-type natriuretic peptide (CNP) when treatments were imposed before in vitro maturation (IVM). Immature oocytes were either directly matured in vitro for 24 h (Control, no pre-IVM), or cultured in basic medium not supplemented or supplemented with CNP (100 nM) (Control pre-IVM and CNP pre-IVM, respectively) for 6 h, followed by IVM for 24 h. The results indicated treatment with CNP before IVM affected patterns of distribution of mitochondria, increased the mitochondrial content, membrane potential, and decreased the ROS content in cattle oocytes before and after IVM. Furthermore, treatment of immature cattle oocytes with CNP before IVM induced marked increases in the relative abundance of mRNA transcripts and proteins related to mitochondria development and antioxidative defense mechanisms. Treatment with CNP before oocyte IVM also resulted in an enhanced relative abundance of sirtuin-1 (SIRT1) mRNA transcript in cattle oocytes. Taken together, these results provide evidence that treatment of cattle oocytes with CNP before IVM improved mitochondrial function and antioxidant defense mechanisms in cattle oocytes. Findings in the present study provide insights into the potential mechanisms by which CNP has positive effects on oocyte cytoplasmic organelles, specifically mitochondria.

C-type natriuretic peptide moderates titin-based cardiomyocyte stiffness.

Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B-KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B-KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.

Effects of C-type natriuretic peptide on meiotic arrest and developmental competence of bovine oocyte derived from small and medium follicles.

The present study aimed to evaluate the effects of C-type natriuretic peptide (CNP) on meiotic arrest and developmental competence of bovine oocyte derived from follicles of different sizes. Collected immature cumulus-oocyte complexes from small follicles (< 3 mm) and medium follicles (3-8 mm) were cultured for 6 h in basal medium supplementated without or with 200 nM CNP. We observed that CNP effectively sustained meiotic arrest at germinal vesicle stage in in vitro cultured bovine oocytes from follicles of different sizes. Moreover, CNP treatment significantly improved the levels of cGMP in both cumulus cells and oocytes, as well as the levels of cAMP in oocytes regardless of follicle size. Based on the above results, we tested the effect of a novel in vitro maturation (IVM) system based on CNP-pretreatment, including a pre-IVM phase for 6 h using 200 nM CNP, followed by a extended IVM phase for 28 h, on developmental competence of bovine oocyte derived from small follicles (< 3 mm) and medium follicles (3-8 mm) compared to standard IVM system. The results showed that athough the novel IVM system based on CNP-pretreatment enhanced the developmental potencial of oocytes obtained from large follicles, but had no effect on the developmental comptence of oocytes obtained from small follicles.

A role for tubular Na+/H+ exchanger NHE3 in the natriuretic effect of the SGLT2 inhibitor empagliflozin.

Inhibitors of proximal tubular Na+-glucose cotransporter 2 (SGLT2) are natriuretic, and they lower blood pressure. There are reports that the activities of SGLT2 and Na+-H+ exchanger 3 (NHE3) are coordinated. If so, then part of the natriuretic response to an SGLT2 inhibitor is mediated by suppressing NHE3. To examine this further, we compared the effects of an SGLT2 inhibitor, empagliflozin, on urine composition and systolic blood pressure (SBP) in nondiabetic mice with tubule-specific NHE3 knockdown (NHE3-ko) and wild-type (WT) littermates. A single dose of empagliflozin, titrated to cause minimal glucosuria, increased urinary excretion of Na+ and bicarbonate and raised urine pH in WT mice but not in NHE3-ko mice. Chronic empagliflozin treatment tended to lower SBP despite higher renal renin mRNA expression and lowered the ratio of SBP to renin mRNA, indicating volume loss. This effect of empagliflozin depended on tubular NHE3. In diabetic Akita mice, chronic empagliflozin enhanced phosphorylation of NHE3 (S552/S605), changes previously linked to lesser NHE3-mediated reabsorption. Chronic empagliflozin also increased expression of genes involved with renal gluconeogenesis, bicarbonate regeneration, and ammonium formation. While this could reflect compensatory responses to acidification of proximal tubular cells resulting from reduced NHE3 activity, these effects were at least in part independent of tubular NHE3 and potentially indicated metabolic adaptations to urinary glucose loss. Moreover, empagliflozin increased luminal α-ketoglutarate, which may serve to stimulate compensatory distal NaCl reabsorption, while cogenerated and excreted ammonium balances urine losses of this "potential bicarbonate." The data implicate NHE3 as a determinant of the natriuretic effect of empagliflozin.

Mechanism of Natriuretic Effect of Oxytocin.

In rats, intramuscular injection of oxytocin (0.25 nmol/100 g body weight) increased sodium excretion from 19±5 to 120±11 µmol/min. A significant correlation (p<0.001) was revealed between renal excretion of oxytocin and sodium ions. Under the action of oxytocin, natriuresis was characterized by diminished reabsorption of fluid in the proximal tubule of the nephron attested by elevated lithium clearance rate and from stimulation of V1a receptors in the cells of thick ascending loop of Henle. Pmp-Tyr(Me)-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2, a V1a receptors antagonist, prevented the natriuretic effect of oxytocin.

Effects of oxytocin administration on the hydromineral balance of median eminence-lesioned rats.

In the clinical setting, acute injuries in hypothalamic mediobasal regions, along with polydipsia and polyuria, have been observed in patients with cerebral salt wasting (CSW). CSW is also characterised by hypovolaemia and hyponatraemia as a result of an early increase in natriuretic peptide activity. Salt and additional amounts of fluid are the main treatment for this disorder. Similarly, experimental lesions to these brain regions, which include the median eminence (ME), produce a well-documented neurological model of polydipsia and polyuria in rats, which is preceded by an early sodium excretion of unknown cause. In the present study, oxytocin (OT) was used to increase the renal sodium loss and prolong the hydroelectrolyte abnormalities of ME-lesioned animals during the first few hours post-surgery. The objective was to determine whether OT-treated ME-lesioned animals increase their sodium appetite and water intake to restore the volume and composition of extracellular body fluid. Electrolytic lesion of the ME increased water intake, urinary volume and sodium excretion of food-deprived rats and also decreased urine osmolality and estimated plasma sodium concentration. OT administration at 8 hours post-surgery reduced water intake, urine output and plasma sodium concentration and also increased urine osmolality and urine sodium excretion between 8 and 24 hours post-lesion. From 24 to 30 hours, more water and hypertonic NaCl was consumed by OT-treated ME-lesioned rats than by physiological saline-treated-ME-lesioned animals. Food availability from 30 to 48 hours reduced the intake of hypertonic saline solution by ME/OT animals, which increased their water and food intake during this period. OT administration therefore appears to enhance the natriuretic effect of ME lesion, producing hydroelectrolyte changes that reduce the water intake of food-deprived animals. Conversely, the presence of hypertonic NaCl increases the fluid intake of these animals, possibly as a result of the plasma sodium depletion and hypovolaemic states previously generated. Finally, the subsequent increase in food intake by ME/OT animals reduces their need for hypertonic NaCl but not water, possibly in response to osmotic thirst. These results are discussed in relation to a possible transient activation of the ME with the consequent secretion of natriuretic peptides stored in terminal swellings, which would be augmented by OT administration. Electrolytic lesion of the ME may therefore represent a useful neurobiological model of CSW.

Chronic treatment with C-type natriuretic peptide impacts differently in the aorta of normotensive and hypertensive rats.

The aim of this study was to determine whether exogenous administration of C-type natriuretic peptide (CNP) induces functional and morphological vascular changes in spontaneously hypertensive rats (SHR) compared with normotensive rats. Male 12-week-old normotensive Wistar and SHR were administered with saline (NaCl 0.9%) or CNP (0.75 µg/h/100 g) for 14 days (subcutaneous micro-osmotic pumps). Systolic blood pressure (SBP) was measured in awake animals and renal parameters were evaluated. After decapitation, the aorta was removed, and vascular morphology, profibrotic markers, and vascular reactivity were measured. In addition, nitric oxide (NO) system and oxidative stress were evaluated. After 14-days of treatment, CNP effectively reduced SBP in SHR without changes in renal function. CNP attenuated vascular remodeling in hypertensive rats, diminishing both profibrotic and pro-inflammatory cytokines. Also, CNP activated the vascular NO system and exerted an antioxidant effect in aortic tissue of both groups, diminishing superoxide production and thiobarbituric acid-reactive substances, and increasing glutathione content. These results show that chronic treatment with CNP attenuates the vascular damage development in a model of essential hypertension, inducing changes in fibrotic, inflammatory, oxidative, and NO pathways that could contribute to beneficial long-term effects on vascular morphology, extracellular matrix composition, and function. The knowledge of these effects of CNP could lead to improved therapeutic strategies to not only control BP but also reduce vascular damage, primarily responsible for the risk of cardiovascular events.

Angiotensin-(1-7) inhibits sodium transport via Mas receptor by increasing nitric oxide production in thick ascending limb.

Sodium transport in the thick ascending loop of Henle (TAL) is tightly regulated by numerous factors, especially angiotensin II (Ang II), a key end-product of the renin-angiotensin system (RAS). However, an alternative end-product of the RAS, angiotensin-(1-7) [Ang-(1-7)], may counter some of the Ang II actions. Indeed, it causes vasodilation and promotes natriuresis through its effects in the proximal and distal tubule. However, its effects on the TAL are unknown. Because the TAL expresses the Mas receptor, an Ang-(1-7) ligand, which in turn may increase NO and inhibit Na+ transport, we hypothesized that Ang-(1-7) inhibits Na transport in the TAL, via a Mas receptor/NO-dependent mechanism. We tested this by measuring transport-dependent oxygen consumption (VO2 ) in TAL suspensions. Administering Ang-(1-7) decreased VO2 ; an effect prevented by dimethyl amiloride and furosemide, signifying that Ang-(1-7) inhibits transport-dependent VO2 in TAL. Ang-(1-7) also increased NO levels, known inhibitors of Na+ transport in the TAL. The effects of Ang-(1-7) on VO2 , as well as on NO levels, were ameliorated by the Mas receptor antagonist, D-Ala, in effect suggesting that Ang-(1-7) may inhibit transport-dependent VO2 in TAL via Mas receptor-dependent activation of the NO pathway. Indeed, blocking NO synthesis with L-NAME prevented the inhibitory actions of Ang-(1-7) on VO2 . Our data suggest that Ang-(1-7) may modulate TAL Na+ transport via Mas receptor-dependent increases in NO leading to the inhibition of transport activity.

Effect of pre-maturation with C-type natriuretic peptide and 3-isobutyl-1-methylxanthine on cumulus-oocyte communication and oocyte developmental competence in cattle.

In vitro embryo production depends on oocyte competence, which is acquired during folliculogenesis, involving cytoplasmic and nuclear processes. In vitro maturation (IVM) induces spontaneous resumption of meiosis, preventing full competence acquisition. The incorporation of a pre-IVM phase with supplementation with C-type natriuretic peptide (CNP) and 3-Isobutyl-1-methylxanthine (IBMX) was used with the aim of improving developmental competence of cattle oocytes. In a preliminary experiment, COCs were cultured with increasing CNP concentrations and nuclear stage assessment was performed. Supplementation with both 100 and 200 nM CNP resulted in more germinal vesicle (GV) arrest at 6 h of culture than those in the control group (79.3%, 76.4% and 59.2%, respectively). In a second experiment, use of 100 nM CNP plus 500 µM IBMX resulted in retention of more oocytes in the GV stage (92.0%) at 6 h of culture compared to supplementation with either CNP or IBMX alone (74.8% and 86.7%, respectively). A subsequent assessment of the effect of the pre-IVM system (6-h of culture with CNP plus IBMX), followed by 20-h of IVM, with comparison to the control at 24-h of IVM was performed. Blastocyst development rate was greater after the pre-IVM phase (45.1% compared with 34.5%). The inclusion of the pre-IVM phase also resulted in an enhanced mitochondrial activity in matured oocytes and sustained integrity of transzonal projections for longer after IVM. In conclusion, CNP and IBMX function synergistically to arrest meiosis in cattle oocytes during a pre-IVM phase, which improves cumulus-oocyte communication and embryo development.

Preclinical evaluation of the diuretic and saluretic effects of (-)-epicatechin and the result of its combination with standard diuretics.

Several studies have suggested that (-)-epicatechin-containing foods and plant extracts benefit conditions that affect the cardiovascular system, such as hypertension and endothelial dysfunction. However, no study was conducted so far to evaluate the potential of this flavonoid on diuretic activity assay. For that, female Wistar normotensive (NTR) and spontaneously hypertensive rats (SHR) received a single oral treatment with (-)-epicatechin (EPI), hydrochlorothiazide (HCTZ) or just vehicle (VEH). The effects of EPI in combination with diuretics for clinical use, as well as with L-NAME, atropine and indomethacin were also explored. Cumulative urine volume, plasma and urinary parameters were evaluated at the end of 8 h experiment. When given to NTR and SHR, at doses of 0.3, 1 and 3 mg/kg, EPI was able to stimulate both diuresis and saluresis (Na+, K+ and Cl-), without interfering with plasma electrolyte content or urinary pH and uric acid values, when compared with VEH-treated only rats. The combination with HCTZ, but not with furosemide or amiloride, successfully strengthened EPI-induced diuresis. This effect was not accompanied by a potentiation of the saluretic effects. On the other hand, when given EPI in combination with amiloride, a significant increase in Cl- excretion and maintenance of the potassium-sparing effects characteristic of this class of diuretics were detected. In addition, the diuretic effect of EPI was enhanced after pretreatment with L-NAME and its action was significantly precluded in the presence of indomethacin, a cyclooxygenase inhibitor. In conclusion, this study shows the diuretic and saluretic properties of EPI in rats, adding another biological activity whose effect may contribute to the different positive actions already described.

[Atrial natriuretic hormones and metabolic syndrome: recent advances]. / Hormones natriurétiques et syndrome métabolique : mise au point.

Natriuretic peptides are a group of hormones including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C type (CNP), urodilatin and guanilyn. ANP (half-life: 2-4 min), is secreted by the atrium, BNP (half-life: 20 min) by the ventricle, CNP by the vascular endothelium, urodilatin by the kidney and guanylin by the intestine. These natriuretic peptides prevent water and salt retention through renal action, vasodilatation and hormonal inhibition of aldosterone, vasopressin and cortisol. These peptides also have a recently demonstrated metabolic effect through an increase of lipolysis, thermogenesis, beta cell proliferation and muscular sensitivity to insulin. Blood levels of these natriuretic peptides depend on "active NPR-A receptors/clearance NPR-C receptors", the last ones being abundant on adipocytes. Therefore, natriuretic peptides act as adipose tissue regulator and constitute a link between blood pressure and metabolic syndrome. They are used as markers and treatment of cardiac failure. Other applications are on going. BNP and NT-proBNP (inactive portion de la pro-hormone) are used as markers of cardiac failure since they have a longer half-life than ANP. BNP decrease is quicker and more important than that one of NT-ProBNP in case of improvement of cardiac failure. Chronic renal insufficiency and beta-blockers increase BNP levels. BNP measurement is useless under treatment with neprilysine inhibitors such as sacubitril, one of the neutral endopeptidases involved in catabolism of natriuretic peptides. The association sacubitril/valsartan is a new treatment of chronic cardiac failure, acting through the decrease of ANP catabolism.

Adipocyte-specific expression of C-type natriuretic peptide suppresses lipid metabolism and adipocyte hypertrophy in adipose tissues in mice fed high-fat diet.

C-type natriuretic peptide (CNP) is expressed in diverse tissues, including adipose and endothelium, and exerts its effects by binding to and activating its receptor, guanylyl cyclase B. Natriuretic peptides regulate intracellular cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP). We recently revealed that overexpression of CNP in endothelial cells protects against high-fat diet (HFD)-induced obesity in mice. Given that endothelial CNP affects adipose tissue during obesity, CNP in adipocytes might directly regulate adipocyte function during obesity. Therefore, to elucidate the effect of CNP in adipocytes, we assessed 3T3-L1 adipocytes and transgenic (Tg) mice that overexpressed CNP specifically in adipocytes (A-CNP). We found that CNP activates the cGMP-VASP pathway in 3T3-L1 adipocytes. Compared with Wt mice, A-CNP Tg mice showed decreases in fat weight and adipocyte hypertrophy and increases in fatty acid ß-oxidation, lipolysis-related gene expression, and energy expenditure during HFD-induced obesity. These effects led to decreased levels of the macrophage marker F4/80 in the mesenteric fat pad and reduced inflammation. Furthermore, A-CNP Tg mice showed improved glucose tolerance and insulin sensitivity, which were associated with enhanced insulin-stimulated Akt phosphorylation. Our results suggest that CNP overexpression in adipocytes protects against adipocyte hypertrophy, excess lipid metabolism, inflammation, and decreased insulin sensitivity during HFD-induced obesity.

Supplementing in vitro embryo production media by NPPC and sildenafil affect the cytoplasmic lipid content and gene expression of bovine cumulus-oocyte complexes and embryos.

In our study, we added natriuretic peptide type C (NPPC) and/or sildenafil during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) followed by in vitro culture (IVC) of embryos with or without sildenafil. We evaluated the effects on the lipid content (LC) of oocytes and embryos and also verified the expression of 96 transcripts related to competence in matured COCs and 96 transcripts related to embryo quality in blastocysts. After IVM, LC was decreased in oocytes by NPPC while sildenafil did not affect LC in oocytes. The genes involved in lipid metabolism and lipid accumulation (DGAT1, PLIN2and PLIN3) were not affected in COCs after treatment during IVM, although the expression of PTX3 (a cumulus cells expansion biomarker) was increased and the hatched blastocyst rate was increased by NPPC during IVM. During IVM, sildenafil increased the mRNA relative abundance of HSF1 and PAF1 and decreased REST in blastocysts. The use of sildenafil in IVC increased the LC of blastocysts. The mRNA abundance in blastocysts produced during IVC with sildenafil was changed for ATF4, XBP1, DNMT3A, DNMT3B, COX2, and SOX2. Although NPPC reduced the LC of oocytes after IVM and upregulated markers for cumulus expansion, embryo production was not affected and the produced blastocysts were able to regain their LC after IVC. Finally, the use of sildenafil during IVC increased the cytoplasmic LC of embryos but did not affect embryo quality, as measured by analysis of 96 transcripts related to embryo quality.

Diuretic and natriuretic effect of luteolin in normotensive and hypertensive rats: Role of muscarinic acetylcholine receptors.

BACKGROUND: Luteolin is a very common phenolic compound found in a wide variety of natural products. Although several biological properties have already been described regarding the beneficial effect of luteolin in the cardiovascular and renal system, no scientific research explored its potential effect as a diuretic agent in experimental trials. METHODS: Groups of male normotensive (NTR) and spontaneously hypertensive rats (SHR) were orally treated with vehicle, hydrochlorothiazide or luteolin. In another experimental set, in order to verify the possible mechanisms of luteolin-induced diuresis, NTR were treated with vehicle, hydrochlorothiazide, amiloride, L-NAME, indomethacin or atropine, 1h before receiving luteolin. The cumulative urine volume, electrolytes excretion, pH and osmolality were measured at the end of the experiment (after 8h). RESULTS: Luteolin, at dose of 3mg/kg, was able to induce both diuretic and natriuretic effect in NTR and SHR groups, without interfering with urinary pH, K+ or Cl- levels. While Ca2+ content remained unchanged in urine from luteolin-treated group of NTR, luteolin reduced Ca2+ excretion in urine from SHR. The treatment with HCTZ and amiloride intensified luteolin-induced diuresis and natriuresis, with an interesting potassium-sparing effect, in addition to an increase in urinary osmolality levels. Moreover, the diuretic and natriuretic action of luteolin was fully avoided in the presence of atropine, a non-selective muscarinic receptor antagonist. CONCLUSIONS: This study revealed the diuretic and natriuretic effect of luteolin in normotensive and hypertensive rats. Our data also showed the involvement of muscarinic acetylcholine receptors for the renal effects of luteolin.

An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield.

STUDY QUESTION: Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? SUMMARY ANSWER: A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. WHAT IS KNOWN ALREADY: IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. STUDY DESIGN, SIZE, DURATION: A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. MAIN RESULTS AND THE ROLE OF CHANCE: PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. LIMITATIONS REASONS FOR CAUTION: The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. STUDY FUNDING/COMPETING INTEREST(S): IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.

Second messenger signaling mechanisms of the brown adipocyte thermogenic program: an integrative perspective.

ß-adrenergic receptors (ßARs) are well established for conveying the signal from catecholamines to adipocytes. Acting through the second messenger cyclic adenosine monophosphate (cAMP) they stimulate lipolysis and also increase the activity of brown adipocytes and the 'browning' of adipocytes within white fat depots (so-called 'brite' or 'beige' adipocytes). Brown adipose tissue mitochondria are enriched with uncoupling protein 1 (UCP1), which is a regulated proton channel that allows the dissipation of chemical energy in the form of heat. The discovery of functional brown adipocytes in humans and inducible brown-like ('beige' or 'brite') adipocytes in rodents have suggested that recruitment and activation of these thermogenic adipocytes could be a promising strategy to increase energy expenditure for obesity therapy. More recently, the cardiac natriuretic peptides and their second messenger cyclic guanosine monophosphate (cGMP) have gained attention as a parallel signaling pathway in adipocytes, with some unique features. In this review, we begin with some important historical work that touches upon the regulation of brown adipocyte development and physiology. We then provide a synopsis of some recent advances in the signaling cascades from ß-adrenergic agonists and natriuretic peptides to drive thermogenic gene expression in the adipocytes and how these two pathways converge at a number of unexpected points. Finally, moving from the physiologic hormonal signaling, we discuss yet another level of control downstream of these signals: the growing appreciation of the emerging roles of non-coding RNAs as important regulators of brown adipocyte formation and function. In this review, we discuss new developments in our understanding of the signaling mechanisms and factors including new secreted proteins and novel non-coding RNAs that control the function as well as the plasticity of the brown/beige adipose tissue as it responds to the energy needs and environmental conditions of the organism.