Intestinal Lymphatic Dysfunction in Kidney Disease.

Kidney disease is associated with adverse consequences in many organs beyond the kidney, including the heart, lungs, brain, and intestines. The kidney-intestinal cross talk involves intestinal epithelial damage, dysbiosis, and generation of uremic toxins. Recent studies reveal that kidney injury expands the intestinal lymphatics, increases lymphatic flow, and alters the composition of mesenteric lymph. The intestinal lymphatics, like blood vessels, are a route for transporting potentially harmful substances generated by the intestines. The lymphatic architecture and actions are uniquely suited to take up and transport large macromolecules, functions that differentiate them from blood vessels, allowing them to play a distinct role in a variety of physiological and pathological processes. Here, we focus on the mechanisms by which kidney diseases result in deleterious changes in intestinal lymphatics and consider a novel paradigm of a vicious cycle of detrimental organ cross talk. This concept involves kidney injury-induced modulation of intestinal lymphatics that promotes production and distribution of harmful factors, which in turn contributes to disease progression in distant organ systems.

Discovery and Characterization of VU0542270, the First Selective Inhibitor of Vascular Kir6.1/SUR2B KATP Channels.

Vascular smooth muscle KATP channels critically regulate blood flow and blood pressure by modulating vascular tone and therefore represent attractive drug targets for treating several cardiovascular disorders. However, the lack of potent inhibitors that can selectively inhibit Kir6.1/SUR2B (vascular KATP) over Kir6.2/SUR1 (pancreatic KATP) has eluded discovery despite decades of intensive research. We therefore screened 47,872 chemically diverse compounds for novel inhibitors of heterologously expressed Kir6.1/SUR2B channels. The most potent inhibitor identified in the screen was an N-aryl-N'-benzyl urea compound termed VU0542270. VU0542270 inhibits Kir6.1/SUR2B with an IC50 of approximately 100 nM but has no apparent activity toward Kir6.2/SUR1 or several other members of the Kir channel family at doses up to 30 µM (>300-fold selectivity). By expressing different combinations of Kir6.1 or Kir6.2 with SUR1, SUR2A, or SUR2B, the VU0542270 binding site was localized to SUR2. Initial structure-activity relationship exploration around VU0542270 revealed basic texture related to structural elements that are required for Kir6.1/SUR2B inhibition. Analysis of the pharmacokinetic properties of VU0542270 showed that it has a short in vivo half-life due to extensive metabolism. In pressure myography experiments on isolated mouse ductus arteriosus vessels, VU0542270 induced ductus arteriosus constriction in a dose-dependent manner similar to that of the nonspecific KATP channel inhibitor glibenclamide. The discovery of VU0542270 provides conceptual proof that SUR2-specific KATP channel inhibitors can be developed using a molecular target-based approach and offers hope for developing cardiovascular therapeutics targeting Kir6.1/SUR2B. SIGNIFICANCE STATEMENT: Small-molecule inhibitors of vascular smooth muscle KATP channels might represent novel therapeutics for patent ductus arteriosus, migraine headache, and sepsis; however, the lack of selective channel inhibitors has slowed progress in these therapeutic areas. Here, this study describes the discovery and characterization of the first vascular-specific KATP channel inhibitor, VU0542270.

A novel role for PGE2-EP4 in the developmental programming of the mouse ductus arteriosus: consequences for vessel maturation and function.

The ductus arteriosus (DA) is a vascular shunt that allows oxygenated blood to bypass the developing lungs in utero. Fetal DA patency requires vasodilatory signaling via the prostaglandin E2 (PGE2) receptor EP4. However, in humans and mice, disrupted PGE2-EP4 signaling in utero causes unexpected patency of the DA (PDA) after birth, suggesting another role for EP4 during development. We used EP4-knockout (KO) mice and acute versus chronic pharmacological approaches to investigate EP4 signaling in DA development and function. Expression analyses identified EP4 as the primary EP receptor in the DA from midgestation to term; inhibitor studies verified EP4 as the primary dilator during this period. Chronic antagonism recapitulated the EP4 KO phenotype and revealed a narrow developmental window when EP4 stimulation is required for postnatal DA closure. Myography studies indicate that despite reduced contractile properties, the EP4 KO DA maintains an intact oxygen response. In newborns, hyperoxia constricted the EP4 KO DA but survival was not improved, and permanent remodeling was disrupted. Vasomotion and increased nitric oxide (NO) sensitivity in the EP4 KO DA suggest incomplete DA development. Analysis of DA maturity markers confirmed a partially immature EP4 KO DA phenotype. Together, our data suggest that EP4 signaling in late gestation plays a key developmental role in establishing a functional term DA. When disrupted in EP4 KO mice, the postnatal DA exhibits signaling and contractile properties characteristic of an immature DA, including impairments in the first, muscular phase of DA closure, in addition to known abnormalities in the second permanent remodeling phase.NEW & NOTEWORTHY EP4 is the primary EP receptor in the ductus arteriosus (DA) and is critical during late gestation for its development and eventual closure. The "paradoxical" patent DA (PDA) phenotype of EP4-knockout mice arises from a combination of impaired contractile potential, altered signaling properties, and a failure to remodel associated with an underdeveloped immature vessel. These findings provide new mechanistic insights into women who receive NSAIDs to treat preterm labor, whose infants have unexplained PDA.

Bronchopulmonary dysplasia is associated with polyhydramnios in a scan for novel perinatal risk factors.

BACKGROUND: The pathogenesis of bronchopulmonary dysplasia (BPD) is multifactorial, and there are limited data about prenatal exposures and risk of BPD. STUDY DESIGN: Our study performed parallel analyses using a logistic regression model in a cohort of 4527 infants with data from a curated registry and using a phenome wide association study (PheWAS) based on ICD9/10-based phecodes. We examined 20 prenatal exposures from a neonatal intensive care unit (NICU) curated registry database related to pregnancy and maternal health as well as 94 maternal diagnosis phecodes with a PheWAS analysis. RESULT: In both the curated registry and PheWAS analyses, polyhydramnios was associated with an increased risk of BPD (OR 5.70, 95% CI 2.78-11.44, p = 1.37 × 10-6). CONCLUSION: Our data suggest that polyhydramnios may be a clinical indicator of premature infants at increased risk for bronchopulmonary dysplasia. Combining curated registry data with PheWAS analysis creates a valuable tool to generate hypotheses. IMPACT: Polyhydramnios was significantly associated with bronchopulmonary dysplasia in both a curated registry and by ICD coding analysis with a phenome wide association study (PheWAS). Preterm polyhydramnios may be a clinical indicator of infants at increased risk for developing bronchopulmonary dysplasia after preterm birth. Combining curated registry with PheWAS analysis creates a valuable tool to generate hypotheses about perinatal risk factors and morbidities associated with preterm birth.

Inflammation, Lymphatics, and Cardiovascular Disease: Amplification by Chronic Kidney Disease.

PURPOSE OF REVIEW: Kidney disease is a strong modulator of the composition and metabolism of the intestinal microbiome that produces toxins and inflammatory factors. The primary pathways for these harmful factors are blood vessels and nerves. Although lymphatic vessels are responsible for clearance of interstitial fluids, macromolecules, and cells, little is known about whether and how kidney injury impacts the intestinal lymphatic network. RECENT FINDINGS: Kidney injury stimulates intestinal lymphangiogenesis, activates lymphatic endothelial cells, and increases mesenteric lymph flow. The mesenteric lymph of kidney-injured animals contains increased levels of cytokines, immune cells, isolevuglandin (IsoLG), a highly reactive dicarbonyl, and of apolipoprotein AI (apoAI). IsoLG is increased in the ileum of kidney injured animals, and intestinal epithelial cells exposed to myeloperoxidase produce more IsoLG. IsoLG-modified apoAI directly increases lymphatic vessel contractions and activates lymphatic endothelial cells. Inhibition of IsoLG by carbonyl scavenger treatment reduces intestinal lymphangiogenesis in kidney-injured animals. Research from our group and others suggests a novel mediator (IsoLG-modified apoAI) and a new pathway (intestinal lymphatic network) in the cross talk between kidneys and intestines and heart. Kidney injury activates intestinal lymphangiogenesis and increases lymphatic flow via mechanisms involving intestinally generated IsoLG. The data identify a new pathway in the kidney gut-heart axis and present a new target for kidney disease-induced intestinal disruptions that may lessen the major adverse consequence of kidney impairment, namely cardiovascular disease.

Kidney Injury Causes Accumulation of Renal Sodium That Modulates Renal Lymphatic Dynamics.

Lymphatic vessels are highly responsive to changes in the interstitial environment. Previously, we showed renal lymphatics express the Na-K-2Cl cotransporter. Since interstitial sodium retention is a hallmark of proteinuric injury, we examined whether renal sodium affects NKCC1 expression and the dynamic pumping function of renal lymphatic vessels. Puromycin aminonucleoside (PAN)-injected rats served as a model of proteinuric kidney injury. Sodium 23Na/1H-MRI was used to measure renal sodium and water content in live animals. Renal lymph, which reflects the interstitial composition, was collected, and the sodium analyzed. The contractile dynamics of isolated renal lymphatic vessels were studied in a perfusion chamber. Cultured lymphatic endothelial cells (LECs) were used to assess direct sodium effects on NKCC1. MRI showed elevation in renal sodium and water in PAN. In addition, renal lymph contained higher sodium, although the plasma sodium showed no difference between PAN and controls. High sodium decreased contractility of renal collecting lymphatic vessels. In LECs, high sodium reduced phosphorylated NKCC1 and SPAK, an upstream activating kinase of NKCC1, and eNOS, a downstream effector of lymphatic contractility. The NKCC1 inhibitor furosemide showed a weaker effect on ejection fraction in isolated renal lymphatics of PAN vs controls. High sodium within the renal interstitium following proteinuric injury is associated with impaired renal lymphatic pumping that may, in part, involve the SPAK-NKCC1-eNOS pathway, which may contribute to sodium retention and reduce lymphatic responsiveness to furosemide. We propose that this lymphatic vessel dysfunction is a novel mechanism of impaired interstitial clearance and edema in proteinuric kidney disease.

Kidney injury-mediated disruption of intestinal lymphatics involves dicarbonyl-modified lipoproteins.

Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized lipids/lipoproteins, little is known about how kidney injury impacts the intestinal lymphatic network, or lipoproteins transported therein. To study this, we used puromycin aminoglycoside-treated rats and NEP25 transgenic mice to show that proteinuric injury expanded the intestinal lymphatic network, activated lymphatic endothelial cells and increased mesenteric lymph flow. The lymph was found to contain increased levels of cytokines, immune cells, and isolevuglandin (a highly reactive dicarbonyl) and to have a greater output of apolipoprotein AI. Plasma levels of cytokines and isolevuglandin were not changed. However, isolevuglandin was also increased in the ileum of proteinuric animals, and intestinal epithelial cells exposed to myeloperoxidase produced more isolevuglandin. Apolipoprotein AI modified by isolevuglandin directly increased lymphatic vessel contractions, activated lymphatic endothelial cells, and enhanced the secretion of the lymphangiogenic promoter vascular endothelial growth factor-C by macrophages. Inhibition of isolevuglandin synthesis by a carbonyl scavenger reduced intestinal isolevuglandin adduct level and lymphangiogenesis. Thus, our data reveal a novel mediator, isolevuglandin modified apolipoprotein AI, and uncover intestinal lymphatic network structure and activity as a new pathway in the crosstalk between kidney and intestine that may contribute to the adverse impact of kidney disease on other organs.

Renal lymphatic vessel dynamics.

Similar to other organs, renal lymphatics remove excess fluid, solutes, and macromolecules from the renal interstitium. Given the kidney's unique role in maintaining body fluid homeostasis, renal lymphatics may be critical in this process. However, little is known regarding the pathways involved in renal lymphatic vessel function, and there are no studies on the effects of drugs targeting impaired interstitial clearance, such as diuretics. Using pressure myography, we showed that renal lymphatic collecting vessels are sensitive to changes in transmural pressure and have an optimal range of effective pumping. In addition, they are responsive to vasoactive factors known to regulate tone in other lymphatic vessels including prostaglandin E2 and nitric oxide, and their spontaneous contractility requires Ca2+ and Cl-. We also demonstrated that Na+-K+-2Cl- cotransporter Nkcc1, but not Nkcc2, is expressed in extrarenal lymphatic vessels. Furosemide, a loop diuretic that inhibits Na+-K+-2Cl- cotransporters, induced a dose-dependent dilation in lymphatic vessels and decreased the magnitude and frequency of spontaneous contractions, thereby reducing the ability of these vessels to propel lymph. Ethacrynic acid, another loop diuretic, had no effect on vessel tone. These data represent a significant step forward in our understanding of the mechanisms underlying renal lymphatic vessel function and highlight potential off-target effects of furosemide that may exacerbate fluid accumulation in edema-forming conditions.

Role of dopamine and selective dopamine receptor agonists on mouse ductus arteriosus tone and responsiveness.

BACKGROUND: Indomethacin treatment for patent ductus arteriosus (PDA) is associated with acute kidney injury (AKI). Fenoldopam, a dopamine (DA) DA1-like receptor agonist dilates the renal vasculature and may preserve renal function during indomethacin treatment. However, limited information exists on DA receptor-mediated signaling in the ductus and fenoldopam may prevent ductus closure given its vasodilatory nature. METHODS: DA receptor expression in CD-1 mouse vessels was analyzed by qPCR and immunohistochemistry. Concentration-response curves were established using pressure myography. Pretreatment with SCH23390 (DA1-like receptor antagonist), phentolamine (α -adrenergic receptor antagonist) or indomethacin addressed mechanisms for DA-induced changes. Fenoldopam's effects on postnatal ductus closure were evaluated in vivo. RESULTS: DA1 receptors were expressed equally in ductus and aorta. High-dose DA induced modest vasoconstriction under newborn O2 conditions. Phentolamine inhibited DA-induced constriction, while SCH23390 augmented constriction, consistent with a vasodilatory role for DA1 receptors. Despite this, fenoldopam had little effect on ductus tone nor indomethacin- or O2-induced constriction and did not impair postnatal closure in vivo. CONCLUSION(S): DA receptors are present in the ductus but have limited physiologic effects. DA-induced ductus vasoconstriction is mediated via α-adrenergic pathways. The absence of DA1-mediated impairment of ductus closure supports the study of potential role for fenoldopam during PDA treatment.

Structure-Activity Relationships, Pharmacokinetics, and Pharmacodynamics of the Kir6.2/SUR1-Specific Channel Opener VU0071063.

Glucose-stimulated insulin secretion from pancreatic ß-cells is controlled by ATP-regulated potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The KATP channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular KATP channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening of Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular KATP channels. VU0071063 induces hyperpolarization of ß-cell membrane potential and inhibits insulin secretion more potently than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose-lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain.

Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery.

Novel therapeutic regulators of uterine contractility are needed to manage preterm labor, induce labor and control postpartum hemorrhage. Therefore, we previously developed a high-throughput assay for large-scale screening of small molecular compounds to regulate calcium-mobilization in primary mouse uterine myometrial cells. The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. Molecular and pharmacological assays were used to compare myometrial cells from four sources: primary cells isolated from term pregnant human and murine myometrium, immortalized pregnant human myometrial (PHM-1) cells and immortalized non-pregnant human myometrial (hTERT-HM) cells. In addition, myometrial cells were compared to vascular SMCs. We found that the transcriptome profiles of hTERT-HM and PHM1 cells were most similar (r = 0.93 and 0.90, respectively) to human primary myometrial cells. Comparative transcriptome profiling of primary human myometrial transcriptome and VSMCs revealed 498 upregulated (p ≤ 0.01, log2FC≥1) genes, of which 142 can serve as uterine-selective druggable targets. In the high-throughput Ca2+-assay, PHM1 cells had the most similar response to primary human myometrial cells in OT-induced Ca2+-release (Emax = 195% and 143%, EC50 = 30 nM and 120 nM, respectively), while all sources of myometrial cells showed excellent and similar robustness and reproducibility (Z' = 0.52 to 0.77). After testing a panel of 61 compounds, we found that the stimulatory and inhibitory responses of hTERT-HM cells were highly-correlated (r = 0.94 and 0.95, respectively) to human primary cells. Moreover, ten compounds were identified that displayed uterine-selectivity (≥5-fold Emax or EC50 compared to VSMCs). Collectively, this study found that hTERT-HM cells exhibited the most similarity to primary human myometrial cells and, therefore, is an optimal substitute for large-scale screening to identify novel therapeutic regulators of myometrial contractility. Moreover, VSMCs can serve as an important counter-screening tool to assess uterine-selectivity of targets and drugs given the similarity observed in the transcriptome and response to compounds.

Mouse models of preterm birth: suggested assessment and reporting guidelines.

Preterm birth affects approximately 1 out of every 10 births in the United States, leading to high rates of mortality and long-term negative health consequences. To investigate the mechanisms leading to preterm birth so as to develop prevention strategies, researchers have developed numerous mouse models of preterm birth. However, the lack of standard definitions for preterm birth in mice limits our field's ability to compare models and make inferences about preterm birth in humans. In this review, we discuss numerous mouse preterm birth models, propose guidelines for experiments and reporting, and suggest markers that can be used to assess whether pups are premature or mature. We argue that adoption of these recommendations will enhance the utility of mice as models for preterm birth.

Effects of antenatal betamethasone on preterm human and mouse ductus arteriosus: comparison with baboon data.

BACKGROUND: Although studies involving preterm infants ≤34 weeks gestation report a decreased incidence of patent ductus arteriosus after antenatal betamethasone, studies involving younger gestation infants report conflicting results. METHODS: We used preterm baboons, mice, and humans (≤276/7 weeks gestation) to examine betamethasone's effects on ductus gene expression and constriction both in vitro and in vivo. RESULTS: In mice, betamethasone increased the sensitivity of the premature ductus to the contractile effects of oxygen without altering the effects of other contractile or vasodilatory stimuli. Betamethasone's effects on oxygen sensitivity could be eliminated by inhibiting endogenous prostaglandin/nitric oxide signaling. In mice and baboons, betamethasone increased the expression of several developmentally regulated genes that mediate oxygen-induced constriction (K+ channels) and inhibit vasodilator signaling (phosphodiesterases). In human infants, betamethasone increased the rate of ductus constriction at all gestational ages. However, in infants born ≤256/7 weeks gestation, betamethasone's contractile effects were only apparent when prostaglandin signaling was inhibited, whereas at 26-27 weeks gestation, betamethasone's contractile effects were apparent even in the absence of prostaglandin inhibitors. CONCLUSIONS: We speculate that betamethasone's contractile effects may be mediated through genes that are developmentally regulated. This could explain why betamethasone's effects vary according to the infant's developmental age at birth.

Selective serotonin reuptake inhibitor exposure constricts the mouse ductus arteriosus in utero.

Use of selective serotonin reuptake inhibitors (SSRIs) is common during pregnancy. Fetal exposure to SSRIs is associated with persistent pulmonary hypertension of the newborn (PPHN); however, a direct link between the two has yet to be established. Conversely, it is well known that PPHN can be caused by premature constriction of the ductus arteriosus (DA), a fetal vessel connecting the pulmonary and systemic circulations. We hypothesized that SSRIs could induce in utero DA constriction. Using isolated vessels and whole-animal models, we sought to determine the effects of two commonly prescribed SSRIs, fluoxetine and sertraline, on the fetal mouse DA. Cannulated vessel myography studies demonstrated that SSRIs caused concentration-dependent DA constriction and made vessels less sensitive to prostaglandin-induced dilation. Moreover, in vivo studies showed that SSRI-exposed mice had inappropriate DA constriction in utero. Taken together, these findings establish that SSRIs promote fetal DA constriction and provide a potential mechanism by which SSRIs could contribute to PPHN.

Effects of Advancing Gestation and Non-Caucasian Race on Ductus Arteriosus Gene Expression.

OBJECTIVE: To identify genes affected by advancing gestation and racial/ethnic origin in human ductus arteriosus (DA). STUDY DESIGN: We collected 3 sets of DA tissue (n = 93, n = 89, n = 91; total = 273 fetuses) from second trimester pregnancies. We examined four genes, with DNA polymorphisms that distribute along racial lines, to identify "Caucasian" and "non-Caucasian" DA. We used real time polymerase chain reaction to measure RNA expression of 48 candidate genes involved in functional closure of the DA, and used multivariable regression analyses to examine the relationships between advancing gestation, "non-Caucasian" race, and gene expression. RESULTS: Mature gestation and non-Caucasian race are significant predictors for identifying infants who will close their patent DA when treated with indomethacin. Advancing gestation consistently altered gene expression in pathways involved with oxygen-induced constriction (eg, calcium-channels, potassium-channels, and endothelin signaling), contractile protein maturation, tissue remodeling, and prostaglandin and nitric oxide signaling in all 3 tissue sets. None of the pathways involved with oxygen-induced constriction appeared to be altered in "non-Caucasian" DA. Two genes, SLCO2A1 and NOS3, (involved with prostaglandin reuptake/metabolism and nitric oxide production, respectively) were consistently decreased in "non-Caucasian" DA. CONCLUSIONS: Prostaglandins and nitric oxide are the most important vasodilators opposing DA closure. Indomethacin inhibits prostaglandin production, but not nitric oxide production. Because decreased SLCO2A1 and NOS3 expression can lead to increased prostaglandin and decreased nitric oxide concentrations, we speculate that prostaglandin-mediated vasodilation may play a more dominant role in maintaining the "non-Caucasian" patent DA, making it more likely to close when inhibited by indomethacin.

Embryonic domains of the aorta derived from diverse origins exhibit distinct properties that converge into a common phenotype in the adult.

Vascular smooth muscle cells (VSMCs) are derived from distinct embryonic origins. Vessels originating from differing smooth muscle cell populations have distinct vascular and pathological properties involving calcification, atherosclerosis, and structural defects such as aneurysm and coarctation. We hypothesized that domains within a single vessel, such as the aorta, vary in phenotype based on embryonic origin. Gene profiling and myographic analyses demonstrated that embryonic ascending and descending aortic domains exhibited distinct phenotypes. In vitro analyses demonstrated that VSMCs from each region were dissimilar in terms of cytoskeletal and migratory properties, and retention of different gene expression patterns. Using the same analysis, we found that these same two domains are indistinguishable in the adult vessel. Our data demonstrate that VSMCs from different embryonic origins are functionally distinct in the embryonic mouse, but converge to assume a common phenotype in the aorta of healthy adults. These findings have fundamental implications for aortic development, function and disease progression.

Transcriptional profiling reveals ductus arteriosus-specific genes that regulate vascular tone.

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BKCa) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BKCa channels caused the DA to constrict, while activation caused DA relaxation even in the presence of O2. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BKCa channels, are promising targets. Specifically, BKCa channel agonists like NS1619 maintain DA patency even in the presence of O2 and may be clinically useful.

Aminoglycoside-mediated relaxation of the ductus arteriosus in sepsis-associated PDA.

Sepsis is strongly associated with patency of the ductus arteriosus (PDA) in critically ill newborns. Inflammation and the aminoglycoside antibiotics used to treat neonatal sepsis cause smooth muscle relaxation, but their contribution to PDA is unknown. We examined whether: 1) lipopolysaccharide (LPS) or inflammatory cytokines cause relaxation of the ex vivo mouse DA; 2) the aminoglycosides gentamicin, tobramycin, or amikacin causes DA relaxation; and 3) newborn infants treated with aminoglycosides have an increased risk of symptomatic PDA (sPDA). Changes in fetal mouse DA tone were measured by pressure myography in response to LPS, TNF-α, IFN-γ, macrophage-inflammatory protein 2, IL-15, IL-13, CXC chemokine ligand 12, or three aminoglycosides. A clinical database of inborn patients of all gestations was analyzed for association between sPDA and aminoglycoside treatment. Contrary to expectation, neither LPS nor any of the inflammatory mediators caused DA relaxation. However, each of the aminoglycosides caused concentration-dependent vasodilation in term and preterm mouse DAs. Pretreatment with indomethacin and N-(G)-nitro-L-arginine methyl ester did not prevent gentamicin-induced DA relaxation. Gentamicin-exposed DAs developed less oxygen-induced constriction than unexposed DAs. Among 488,349 infants who met the study criteria, 40,472 (8.3%) had sPDA. Confounder-adjusted odds of sPDA were higher in gentamicin-exposed infants, <25 wk and >32 wk. Together, these findings suggest that factors other than inflammation contribute to PDA. Aminoglycoside-induced vasorelaxation and inhibition of oxygen-induced DA constriction support the paradox that antibiotic treatment of sepsis may contribute to DA relaxation. This association was also found in newborn infants, suggesting that antibiotic selection may be an important consideration in efforts to reduce sepsis-associated PDA.

Efficacy of paracetamol on patent ductus arteriosus closure may be dose dependent: evidence from human and murine studies.

BACKGROUND: We evaluated the clinical effectiveness of variable courses of paracetamol on patent ductus arteriosus (PDA) closure and examined its effect on the in vitro term and preterm murine ductus arteriosus (DA). METHODS: Neonates received one of the following three paracetamol regimens: short course of oral paracetamol (SCOP), long course of oral paracetamol (LCOP), and intravenous paracetamol (IVP) for 2-6 d. Pressure myography was used to examine changes in vasomotor tone of the preterm and term mouse DA in response to paracetamol or indomethacin. Their effect on prostaglandin synthesis by DA explants was measured by mass spectroscopy. RESULTS: Twenty-one preterm infants were included. No changes in PDA hemodynamics were seen in SCOP infants (n = 5). The PDA became less significant and eventually closed in six LCOP infants (n = 7). PDA closure was achieved in eight IVP infants (n = 9). On pressure myograph, paracetamol induced a concentration-dependent constriction of the term mouse DA, up to 30% of baseline (P < 0.01), but required >1 µmol/l. Indomethacin induced greater DA constriction and suppression of prostaglandin synthesis (P < 0.05). CONCLUSION: The clinical efficacy of paracetamol on PDA closure may depend on the duration of treatment and the mode of administration. Paracetamol is less potent than indomethacin for constriction of the mouse DA in vitro.