Network pharmacology-based research into the mechanism of ferulic acid on acute lung injury through enhancing transepithelial sodium transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferulic acid (FA) has shown potential therapeutic applications in treating lung diseases. However, the underlying mechanisms by which FA ameliorates acute lung injury (ALI) have not been distinctly elucidated. AIM OF THE STUDY: The project aims to observe the therapeutic effects of FA on lipopolysaccharide-induced ALI and to elucidate its specific mechanisms in regulating epithelial sodium channel (ENaC), which majors in alveolar fluid clearance during ALI. MATERIALS AND METHODS: In this study, the possible pathways of FA were determined through network pharmacology analyses. The mechanisms of FA in ALI were verified by in vivo mouse model and in vitro studies, including primary alveolar epithelial type 2 cells and three-dimensional alveolar organoid models. RESULTS: FA ameliorated ALI by improving lung pathological changes, reducing pulmonary edema, and upregulating the α/γ-ENaC expression in C57BL/J male mice. Simultaneously, FA was observed to augment ENaC levels in both three-dimensional alveolar organoid and alveolar epithelial type 2 cells models. Network pharmacology techniques and experimental data from inhibition or knockdown of IkappaB kinase ß (IKKß) proved that FA reduced the phosphorylation of IKKß/nuclear factor-kappaB (NF-κB) and eliminated the lipopolysaccharide-inhibited expression of ENaC, which could be regulated by nuclear protein NF-κB p65 directly. CONCLUSIONS: FA could enhance the expression of ENaC at least in part by inhibiting the IKKß/NF-κB signaling pathway, which may potentially pave the way for promising treatment of ALI.

Comprehensive mapping of Epithelial Na+ channel α expression in the mouse brain.

Epithelial sodium channel (ENaC) is responsible for regulating Na+ homeostasis. While its physiological functions have been investigated extensively in peripheral tissues, far fewer studies have explored its functions in the brain. Since our limited knowledge of ENaC's distribution in the brain impedes our understanding of its functions there, we decided to explore the whole-brain expression pattern of the Scnn1a gene, which encodes the core ENaC complex component ENaCα. To visualize Scnn1a expression in the brain, we crossed Scnn1a-Cre mice with Rosa26-lsl-tdTomato mice. Brain sections were subjected to immunofluorescence staining using antibodies against NeuN or Myelin Binding Protein (MBP), followed by the acquisition of confocal images. We observed robust tdTomato fluorescence not only in the soma of cortical layer 4, the thalamus, and a subset of amygdalar nuclei, but also in axonal projections in the hippocampus and striatum. We also observed expression in specific hypothalamic nuclei. Contrary to previous reports, however, we did not detect significant expression in the circumventricular organs, which are known for their role in regulating Na+ balance. Finally, we detected fluorescence in cells lining the ventricles and in the perivascular cells of the median eminence. Our comprehensive mapping of Scnn1a-expressing cells in the brain will provide a solid foundation for further investigations of the physiological roles ENaC plays within the central nervous system.

Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.

Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation.

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

High-salt diet accelerates bone loss accompanied by activation of ion channels related to kidney and bone tissue in ovariectomized rats.

Excessive salt intake can induce a variety of diseases, such as hypertension, cardiovascular disease, kidney disease and so on，it is also one of the factors promoting bone resorption. The mechanism of osteoporosis-induced exacerbations of high salt diet is not well-defined. In this study, we used ovariectomized 6-month-old Sprague Dawley rats to construct a high bone turnover model, and then administrated with high sodium chloride diet (2.0% w/w NaCl, 8.0% w/w NaCl) for 12 weeks to observe the effect of high salt diet on bone metabolism. The results showed that high salt diet could lead to the destruction of bone microstructure, promote the excretion of urinary calcium and phosphorus and accelerate the bone turnover, as well as cause the pathologic structural abnormalities in renal tubular. At the same time, it was accompanied by the up-regulated expression of the epithelial sodium channel (ENaCα), voltage-gated chloride channels (ClC)- 3 and the down-regulated expression of Na-Cl cotransporter (NCC), sodium calcium exchanger (NCX1) in femoral tissue and renal tubules. These findings confirm that high salt diet can destroy the microstructure of bone by increasing bone resorption and affect some ion channels of bone tissue and renal tubule in ovariectomized rats.

Albumin Stimulates Epithelial Na+ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway.

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na+ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na+ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na+ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na+ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na+ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na+ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na+ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway.

ANKRD36 Is Involved in Hypertension by Altering Expression of ENaC Genes.

[Figure: see text].

Is the ENaC Dysregulation in CF an Effect of Protein-Lipid Interaction in the Membranes?

While approximately 2000 mutations have been discovered in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), only a small amount (about 10%) is associated with clinical cystic fibrosis (CF) disease. The discovery of the association between CFTR and the hyperactive epithelial sodium channel (ENaC) has raised the question of the influence of ENaC on the clinical CF phenotype. ENaC disturbance contributes to the pathological secretion, and overexpression of one ENaC subunit, the ß-unit, can give a CF-like phenotype in mice with normal acting CFTR. The development of ENaC channel modulators is now in progress. Both CFTR and ENaC are located in the cell membrane and are influenced by its lipid configuration. Recent studies have emphasized the importance of the interaction of lipids and these proteins in the membranes. Linoleic acid deficiency is the most prevailing lipid abnormality in CF, and linoleic acid is an important constituent of membranes. The influence on sodium excretion by linoleic acid supplementation indicates that lipid-protein interaction is of importance for the clinical pathophysiology in CF. Further studies of this association can imply a simple clinical adjuvant in CF therapy.

The physiological and molecular mechanisms to maintain water and salt homeostasis in response to high salt intake in Mongolian gerbils (Meriones unguiculatus).

Desert rodents are faced with many challenges such as high dietary salt in their natural habitats and they have evolved abilities to conserve water and tolerate salt. However, the physiological and molecular mechanisms involved in water and salt balances in desert rodents are unknown. We hypothesized that desert rodents regulated water and salt balances by altering the expression of AQP2 and α-ENaC in the kidney. Mongolian gerbils (Meriones unguiculatus), a desert species, were acclimated to drinking water with different salt contents: (0, control; 4% NaCl, moderate salt, MS; 8% NaCl, high salt, HS) for 4 weeks. The gerbils drinking salty water had lower body mass, food intake, water intake, metabolic water production and urine volume. The HS gerbils increased the expression of arginine vasopressin (AVP) in the hypothalamus, and also enhanced the expression of AQP2 and cAMP/PKA/CREB signaling pathway in the kidney. In addition, these gerbils reduced serum aldosterone levels and α-ENaC expression in the kidney. Creatinine clearance was lower in the HS group than that in the control group, but serum and urine creatinine levels did not change. These data indicate that desert rodents rely on AVP-dependent upregulation of AQP2 and aldosterone-dependent downregulation of α-ENaC in the kidney to promote water reabsorption and sodium excretion under high salt intake.

Glucocorticoids Reverse Diluted Hyponatremia Through Inhibiting Arginine Vasopressin Pathway in Heart Failure Rats.

Background Arginine vasopressin dependent antidiuresis plays a key role in water-sodium retention in heart failure. In recent years, the role of glucocorticoids in the control of body fluid homeostasis has been extensively investigated. Glucocorticoid deficiency can activate V2R (vasopressin receptor 2), increase aquaporins expression, and result in hyponatremia, all of which can be reversed by glucocorticoid supplement. Methods and Results Heart failure was induced by coronary artery ligation for 8 weeks. A total of 32 rats were randomly assigned to 4 groups (n=8/group): sham surgery group, congestive heart failure group, dexamethasone group, and dexamethasone in combination with glucocorticoid receptor antagonist RU486 group. An acute water loading test was administered 6 hours after drug administration. Left ventricular function was measured by a pressure-volume catheter. Protein expressions were determined by immunohistochemistry and immunoblotting. The pressure-volume loop analysis showed that dexamethasone improves cardiac function in rats with heart failure. Western blotting confirmed that dexamethasone remarkably reduces the expressions of V2R, aquaporin 2, and aquaporin 3 in the renal-collecting ducts. As a result of V2R downregulation, the expressions of glucocorticoid regulated kinase 1, apical epithelial sodium channels, and the furosemide-sensitive Na-K-2Cl cotransporter were also downregulated. These favorable effects induced by dexamethasone were mostly abolished by the glucocorticoid receptor inhibitor RU486, indicating that the aforementioned effects are glucocorticoid receptor mediated. Conclusions Glucocorticoids can reverse diluted hyponatremia via inhibiting the vasopressin receptor pathway in rats with heart failure.

Bu Yang Huan Wu decoction prevents reperfusion injury following ischemic stroke in rats via inhibition of HIF-1 α, VEGF and promotion ß-ENaC expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu Yang Huan Wu Decoction (BYHW) is a famous traditional Chinese medicine (TCM) formula used in China for the treatment of cerebral ischemic stroke. But the protective effects and underlining mechanisms of BYHW remain unclear. AIM OF THE STUDY: This study was designed to investigate the protective effects and underlining signaling mechanisms of BYHW on brain tissues in a rat model of cerebral ischemic reperfusion (I/R) injury. MATERIALS AND METHODS: Liquid chromatography was used to verify the composition of BYHW. The cerebral edema and infarct volume were measured by magnetic resonance imaging (MRI). The morphology and ultrastructure of ischemic penumbra brain tissues were observed by hematoxylin-eosin (HE) and transmission electron microscopy (TEM). The expression levels of HIF-1 α, VEGF and ß-ENaC were tested using immunohistochemistry technique, western blot and quantitative PCR analysis, respectively. RESULTS: Administration of BYHW significantly decreased cerebral edema, rat neurological function scores, reduced brain infarct volume. At the same time, BYHW had protective effect on the blood-brain barrier (BBB), which improved the morphology and ultrastructure of ischemic penumbra brain tissues. BYHW treatment significantly decreased the protein and mRNA levels of HIF-1 α and VEGF compared with the model treatment. In addition, BYHW treatment significantly up-regulated the protein and mRNA levels of ß-ENaC. CONCLUSIONS: BYHW protected against cerebral I/R injury in MCAO rats through inhibiting the activation of the HIF-1 α /VEGF pathway and stabilizing ion channel of ß-ENaC in brain, indicating that BYHW shows potential for stroke treatment in acute stage.

Inhaled Biologicals for the Treatment of Cystic Fibrosis.

BACKGROUND: Cystic Fibrosis (CF), one of the most frequent genetic diseases, is characterized by the production of viscous mucus in several organs. In the lungs, mucus clogs the airways and traps bacteria, leading to recurrent/resistant infections and lung damage. For cystic fibrosis patients, respiratory failure is still lethal in early adulthood since available treatments display incomplete efficacy. OBJECTIVE: The objective of this review is to extend the current knowledge in the field of available treatments for cystic fibrosis. A special focus has been given to inhaled peptide-based drugs. METHODS: The current review is based on recent and/or relevant literature and patents already available in various scientific databases, which include PubMed, PubMed Central, Patentscope and Science Direct. The information obtained through these diverse databases is compiled, critically interpreted and presented in the current study. An in-depth but not systematic approach to the specific research question has been adopted. RESULTS: Recently, peptides have been proposed as possible pharmacologic agents for the treatment of respiratory diseases. Of note, peptides are suitable to be administered by inhalation to maximize efficacy and reduce systemic side effects. Moreover, innovative delivery carriers have been developed for drug administration through inhalation, allowing not only protection against proteolysis, but also a prolonged and controlled release. CONCLUSION: Here, we summarize newly patented peptides that have been developed in the last few years and advanced technologies for inhaled drug delivery to treat cystic fibrosis.

Effect of dietary salt intake on epithelial Na+ channels (ENaCs) in the hypothalamus of Dahl salt-sensitive rats.

All three epithelial Na+ channel (ENaC) subunits (α, ß, and γ) and the mineralocorticoid receptor (MR), a known regulator of ENaC, are located in vasopressin (VP) synthesizing magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. Our previous study showed that ENaC mediates a Na+ leak current that affects the steady-state membrane potential of VP neurons. This study was conducted in Dahl salt-sensitive (Dahl-SS) rats to determine if any abnormal responses in the expression of ENaC subunits and MR occur in the hypothalamus and kidney in response to a high dietary salt intake. After 21 days of high salt consumption, Dahl-SS rat resulted in a significant increase in γENaC expression and exhibited proteolytic cleavage of this subunit compared to Sprague-Dawley (SD) rats. Additionally, Dahl-SS rats had dense somato-dendritic γENaC immunoreactivity in VP neurons, which was absent in SD rats. In contrast, SD rats fed a high salt diet had significantly decreased αENaC subunit expression in the kidney and MR expression in the hypothalamus. Plasma osmolality measured daily for 22 days demonstrated that Dahl-SS rats fed a high salt diet had a steady increase in plasma osmolality, whereas SD rats had an initial increase that decreased to baseline levels. Findings from this study demonstrate that Dahl-SS rats lack a compensatory mechanism to down regulate ENaC during high dietary salt consumption, which may contribute to the development of hypertension.

Compromised regulation of the collecting duct ENaC activity in mice lacking AT1a receptor.

ENaC-mediated sodium reabsorption in the collecting duct (CD) is a critical determinant of urinary sodium excretion. Existing evidence suggest direct stimulatory actions of Angiotensin II (Ang II) on ENaC in the CD, independently of the aldosterone-mineralocorticoid receptor (MR) signaling. Deletion of the major renal AT1 receptor isoform, AT1a R, decreases blood pressure and reduces ENaC abundance despite elevated aldosterone levels. The mechanism of this insufficient compensation is not known. Here, we used patch clamp electrophysiology in freshly isolated split-opened CDs to investigate how AT1a R dysfunction compromises functional ENaC activity and its regulation by dietary salt intake. Ang II had no effect on ENaC activity in CDs from AT1a R -/- mice suggesting no complementary contribution of AT2 receptors. We next found that AT1a R deficient mice had lower ENaC activity when fed with low (<0.01% Na+ ) and regular (0.32% Na+ ) but not with high (∼2% Na+ ) salt diet, when compared to the respective values obtained in Wild type (WT) animals. Inhibition of AT1 R with losartan in wild-type animals reproduces the effects of genetic ablation of AT1a R on ENaC activity arguing against contribution of developmental factors. Interestingly, manipulation with aldosterone-MR signaling via deoxycosterone acetate (DOCA) and spironolactone had much reduced influence on ENaC activity upon AT1a R deletion. Consistently, AT1a R -/- mice have a markedly diminished MR abundance in cytosol. Overall, we conclude that AT1a R deficiency elicits a complex inhibitory effect on ENaC activity by attenuating ENaC Po and precluding adequate compensation via aldosterone cascade due to decreased MR availability.

Short-Term High-Salt Diet Increases Corin Level to Regulate the Salt-Water Balance in Humans and Rodents.

BACKGROUND: Dietary sodium and potassium affect the fluctuation in blood pressure (BP) and renal function. Corin, with its enzymatic activity to convert pro-atrial natriuretic peptide (pro-ANP) to biologically active ANP, regulates BP, cardiac, and renal functions. We investigated whether corin expression responds to a high-salt (HS) diet to regulate salt and water balance. METHODS: Forty-two volunteers followed 3 sequential diets for 7 days each: a low-salt (LS) diet (3.0 g/day NaCl), a HS diet (18.0 g/day NaCl), followed by an HS diet with K+ supplementation (HS + K+) (18.0 g/day NaCl and 4.5 g/day KCl). RESULTS: Corin level was higher with the HS diet than the LS and HS + K+ diets and was positively correlated with systolic BP (SBP) and 24-hour urinary Na+ and microalbumin (U-mALB) excretion. In rodents, serum and renal levels of corin were transiently increased with the HS diet and were decreased if the HS diet was continued for up to 7 days. HS loading increased SBP, 24-hour urinary Na+, U-mALB excretion, and the expression of proprotein convertase subtilisin/kexin-6 (PCSK6), a corin activator. Knockdown of PCSK6 or corin in high salt-treated M1-cortical collecting duct (M1-CCD) cells increased the expression of aquaporin 2 (AQP2) and ß-epithelial Na+ channel (ß-ENaC). CONCLUSIONS: Short-term HS may induce the PCSK6-corin-ANP-AQP2/ß-ENaC pathway in the kidney. Enhanced serum corin level in humans and rodents is positively correlated with HS-induced SBP and 24-hour urinary Na+ and U-mALB excretion, which suggests that corin is involved in the salt-water balance in response to HS intake. CLINICAL TRIALS REGISTRATION: Public Trials Registry Number NCT02915315.

Increased blood pressure in nesfatin/nuclebindin-2-transgenic mice.

Nesfatin/nucleobindin-2 (nesf/NUCB2), a precursor of the anorexigenic protein nesfatin-1, is selectively expressed in the hypothalamic nuclei, which are central to the regulation of the autonomic nervous system. The present study sought to investigate the involvement of nesf/NUCB2 in the regulation of blood pressure and ingestive behavior, by using nesf/NUCB2-transgenic (Tg) mice. Blood pressure and heart rates were measured under conscious and unconscious conditions. Twenty-four-hour water intake and urine volume of male nesf/NUCB2-Tg mice and their littermates in metabolic cages were measured. After killing, kidney weight was measured and the mRNA expression of epithelial sodium channel (ENaC)-α and ENaC-γ was measured in the hypothalamus and kidney with real-time PCR. Systolic, diastolic and mean blood pressure were significantly higher in nesf/NUCB2-Tg mice, but pulse rate was not affected in conscious mice. In contrast, isoflurane anesthesia prevented an increase in blood pressure in the nesf/NUCB2-Tg mice. Twenty-four-hour water intake and urine volume were significantly higher in the nesf/NUCB2-Tg mice than in their non-Tg littermates. Urine sodium concentration was significantly lower in the nesf/NUCB2-Tg mice, although the serum sodium concentration and urine sodium excretion were not different between the genotypes. Kidney weight was significantly higher in the nesf/NUCB2-Tg mice than their non-Tg littermates, although there were no clear differences in the kidney histological findings between genotypes. The mRNA expression of ENaC-γ, but not ENaC-α, was decreased in the hypothalami of nesf/NUCB2-Tg mice. Our data suggested that Nesf/NUCB2 is involved in the regulation of blood pressure in the brain.

Salt supplementation ameliorates developmental kidney defects in COX-2-/- mice.

Deficiency of cyclooxygenase-2 (COX-2) activity in the early postnatal period causes impairment of kidney development leading to kidney insufficiency. We hypothesize that impaired NaCl reabsorption during the first days of life is a substantial cause for nephrogenic defects observed in COX-2-/- mice and that salt supplementation corrects these defects. Daily injections of NaCl (0.8 mg·g-1·day-1) for the first 10 days after birth ameliorated impaired kidney development in COX-2-/- pups resulting in an increase in glomerular size and fewer immature superficial glomeruli. However, impaired renal subcortical growth was not corrected. Increasing renal tubular flow by volume load or injections of KCl did not relieve the renal histomorphological damage. Administration of torsemide and spironolactone also affected nephrogenesis resulting in diminished glomeruli and cortical thinning. Treatment of COX-2-/- pups with NaCl/DOCA caused a stronger mitigation of glomerular size and induced a slight but significant growth of cortical tissue mass. After birth, renal mRNA expression of NHE3, NKCC2, ROMK, NCCT, ENaC, and Na+/K+-ATPase increased relative to postnatal day 2 in wild-type mice. However, in COX-2-/- mice, a significantly lower expression was observed for NCCT, whereas NaCl/DOCA treatment significantly increased NHE3 and ROMK expression. Long-term effects of postnatal NaCl/DOCA injections indicate improved kidney function with normalization of pathologically enhanced creatinine and urea plasma levels; also, albumin excretion was observed. In summary, we present evidence that salt supplementation during the COX-2-dependent time frame of nephrogenesis partly reverses renal morphological defects in COX-2-/- mice and improves kidney function.

Combinatorial effects of quercetin and sex-steroids on fluid and electrolytes' (Na+, Cl-, HCO3-) secretory mechanisms in the uterus of ovariectomised female Sprague-Dawley rats.

Dysregulation of uterine fluid environment could impair successful reproduction and this could be due to the effect of environmental estrogens. Therefore, in this study, effect of quercetin, an environmental estrogen on uterine fluid and electrolytes concentrations were investigated under sex-steroid influence. Ovariectomised adult female Sprague-Dawley rats were given 10, 50 or 100mg/kg/day quercetin subcutaneously with 17-ß estradiol (E) for seven days or three days E, then three days E plus progesterone (P) (E+P) treatment. Uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations were determined by in-vivo perfusion. Following sacrifice, uteri were harvested and levels of the proteins of interest were identified by Western blotting and Realtime PCR. Distribution of these proteins in the uterus was observed by immunofluorescence. Levels of uterine cAMP were measured by enzyme-linked immunoassay (EIA). Administration of quercetin at increasing doses increased uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations, but to the levels lesser than that of E. In concordant, levels of CFTR, SLC4A4, ENaC (α, ß and γ), Na+/K+-ATPase, GPα/ß, AC and cAMP in the uterus increased following increased in the doses of quercetin. Co-administration of quercetin with E caused uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations to decrease. In concordant, uterine CFTR, SLC26A6, SLC4A4, ENaC (α, ß and γ), Na+/K+-ATPase, GPα/ß, AC and cAMP decreased. Greatest effects were observed following co-administration of 10mg/kg/day quercetin with E. Co-administration of quercetin with E+P caused uterine fluid Na+ and HCO3- concentrations to increase but no changes in fluid secretion rate and Cl- concentration were observed. Co-administration of high dose quercetin (100 mg/kg/day) with E+P caused uterine CFTR, SLC26A6, AC, GPα/ß and ENaC (α, ß and γ) to increase. Quercetin-induced changes in the uterine fluid secretion rate and electrolytes concentrations could potentially affect the uterine reproductive functions under female sex-steroid influence.

Actions of Quercetin, a Polyphenol, on Blood Pressure.

Disorder of blood pressure control causes serious diseases in the cardiovascular system. This review focuses on the anti-hypertensive action of quercetin, a flavonoid, which is one of the polyphenols characterized as the compounds containing large multiples of phenol structural units, by varying the values of various blood pressure regulatory factors, such as vascular compliance, peripheral vascular resistance, and total blood volume via anti-inflammatory and anti-oxidant actions. In addition to the anti-inflammatory and anti-oxidant actions of quercetin, we especially describe a novel mechanism of quercetin's action on the cytosolic Cl- concentration ([Cl-]c) and novel roles of the cytosolic Cl- i.e.: (1) quercetin elevates [Cl-]c by activating Na⁺-K⁺-2Cl- cotransporter 1 (NKCC1) in renal epithelial cells contributing to Na⁺ reabsorption via the epithelial Na⁺ channel (ENaC); (2) the quercetin-induced elevation of [Cl-]c in renal epithelial cells diminishes expression of ENaC leading to a decrease in renal Na⁺ reabsorption; and (3) this reduction of ENaC-mediated Na⁺ reabsorption in renal epithelial cells drops volume-dependent elevated blood pressure. In this review, we introduce novel, unique mechanisms of quercetin's anti-hypertensive action via activation of NKCC1 in detail.

Proanthocyanidins block aldosterone-dependent up-regulation of cardiac gamma ENaC and Nedd4-2 inactivation via SGK1.

Aldosterone plays a central role in the development of cardiac pathological states involving ion transport imbalances, especially sodium transport. We have previously demonstrated a cardioprotective effect of proanthocyanidins in aldosterone-treated rats. Our objective was to investigate for the first time the effect of proanthocyanidins on serum and glucocorticoid-regulated kinase 1 (SGK1), epithelial Na+ channel (γ-ENaC), neuronal precursor cells expressed developmentally down-regulated 4-2 (Nedd4-2) and phosphoNedd4-2 protein expression in the hearts of aldosterone-treated rats. Male Wistar rats received aldosterone (1mg kg-1day-1)+1% NaCl for 3weeks. Half of the animals in each group were simultaneously treated with the proanthocyanidins-rich extract (80% w/w) (PRO80, 5mg kg-1day-1). Hypertension and diastolic dysfunction induced by aldosterone were abolished by treatment with PRO80. Expression of fibrotic, inflammatory and oxidative mediators were increased by aldosterone-salt administration and blunted by PRO80. Antioxidant capacity was improved by PRO80. The up-regulated aldosterone mediator SGK1, ENaC and p-Nedd4-2/total Nedd4-2 ratio were blocked by PRO80. PRO80 blunted aldosterone-mineralocorticoid-mediated up-regulation of ENaC provides new mechanistic insight of the beneficial effect of proanthocyanidins preventing the cardiac alterations induced by aldosterone excess.