Increased Fluid Secretion Research Articles

Kaempferol activates chloride secretion via the cAMP/PKA signaling pathway and expression of CFTR in T84 cells.

Kaempferol is a flavonol identified as the most potent activator of chloride (Cl-) secretion among other flavonoids in airway epithelial cells. This study aimed to investigate the cellular mechanisms by which kaempferol stimulates Cl- secretion in the T84 human colon carcinoma cell line by Ussing chambers and voltage clamp technique. Bilateral addition of kaempferol (1-100µM) increased short-circuit current (I sc ) in a concentration-dependent manner. Ion substitution of Cl- or CFTR inhibitors NPPB and glibenclamide or a Na+/K+/2Cl- cotransporter inhibitor bumetanide attenuated kaempferol-induced I sc response. In permeabilized monolayers, selective channel inhibitors CFTRinh-172 and CaCCinh-A01 inhibited kaempferol-induced apical Cl- current (I Cl ), and K+ blockers BaCl2 and clotrimazole inhibited basolateral K+ current (I Kb ). The kaempferol-induced I Cl showed no additive effects with forskolin or 8cpt-cAMP. The kaempferol-induced I Cl was mostly abolished by protein kinase A inhibitor H89, but not by tyrosine kinase inhibitors, AG490 and tyrphostin A23, or tyrosine phosphatase inhibitor vanadate. Treatment with kaempferol for 24h increased the expression of CFTR protein as determined by the Western blot analysis. These results demonstrated that kaempferol activates Cl- secretion across T84 cells by activating the apical Cl- current and basolateral K+ current. The mechanisms may involve the cAMP/PKA pathway and CFTR expression. Taken together, these findings reveal the beneficial effects of kaempferol to increase fluid secretion which can be used to treat constipation.

Neurotensin and xenin stimulates pancreatic exocrine secretion through the peripheral cholinergic nerves in conscious sheep

The present study aimed to clarify the effects of neurotensin and xenin on pancreatic exocrine secretion in conscious sheep and their mechanism of actions. The animals were equipped with two silastic cannulae in the common bile duct to separately collect pancreatic fluid and bile, and a silastic cannula in the proximal duodenum to continuously return the mixed fluids. NT and xenin were intravenously injected at range of 0.01–3.0 nmol/kg during the phase I of duodenal migrating motor complex. A single intravenous NT injection significantly and dose-dependently increased pancreatic fluid, protein, and bicarbonate outputs. The effect of NT at 1 nmol/kg was completely inhibited by a background intravenous infusion of atropine methyl nitrate at a dose of 10 nmol/kg/min, however, the effect was not altered by a prior injection of the neurotensin receptor subtype (NTR)-1 antagonist SR 48692 at 60 nmol/kg. Moreover, a single intravenous xenin-25 injection significantly and dose-dependently increased pancreatic fluid and protein output, whereas the effect of xenin-25 did not clearly show dose-dependence. The prior SR 48692 injection at 30 nmol/kg did not significantly alter the effects of xenin-25 at 0.3 nmol/kg, while the atropine infusion significantly inhibited the increase in fluid secretion. Under the atropine infusion, xenin-25 at 0.3 nmol/kg did not increase protein and bicarbonate outputs, whereas the inhibitory effect of the atropine was not significant compared to that of the single injection of xenin-25. A single intravenous injection of NTR-2 agonist levocabastine at 0.1–3 nmol/kg did not alter pancreatic exocrine secretion. These results suggest that both NT and xenin-25 effectively stimulates pancreatic exocrine secretion through the peripheral cholinergic system in sheep and that NTR-2 is not involved in the regulation of pancreatic exocrine secretion, however, we did not precisely determine the role of NTR-1 in the actions of both the peptides on pancreatic exocrine secretion.

Combined agonists act synergistically to increase mucociliary clearance in a cystic fibrosis airway model

Mucus clearance, a primary innate defense mechanism of airways, is defective in patients with cystic fibrosis (CF) and CF animals. In previous work, the combination of a low dose of the cholinergic agonist, carbachol with forskolin or a β adrenergic agonist, isoproterenol synergistically increased mucociliary clearance velocity (MCCV) in ferret tracheas. Importantly, the present study shows that synergistic MCCV can also be produced in CF ferrets, with increases ~ 55% of WT. Synergistic MCCV was also produced in pigs. The combined agonists increased MCCV by increasing surface fluid via multiple mechanisms: increased fluid secretion from submucosal glands, increased anion secretion across surface epithelia and decreased Na+ absorption. To avoid bronchoconstriction, the cAMP agonist was applied 30 min before carbachol. This approach to increasing mucus clearance warrants testing for safety and efficacy in humans as a potential therapeutic for muco-obstructive diseases.

POS-431 ALTERED REGULATION OF TRYPTOPHAN METABOLISM AND ARYL HYDROCARBON RECEPTOR DISTRIBUTION IN RODENT POLYCYSTIC KIDNEYS

Polycystic kidney disease (PKD) is an inherited disorder characterized by stimulated cell proliferation, increased fluid secretion into cysts, and interstitial inflammation in renal tissue. Many mechanisms of disease progression have been determined in both autosomal dominant and autosomal recessive PKD, but the exact signaling pathways responsible for cystogenesis remain unclear. To explore novel mechanisms of PKD progression, we evaluated altered metabolic activity and associated receptor expression and distribution in the kidneys of 4- and 20-week-old PCK rats, an orthologous model of human autosomal recessive PKD, by mass spectrometry, quantitative analysis, real-time RT-PCR and immunofluorescence. Based on mass spectrometry and quantitative analysis results, the abundances of kynurenine and kynurenic acid were significantly higher in both 4- and 20-week-old PCK kidneys compared with age-matched wild-type kidneys. Since kynurenine and kynurenic acid are endogenous agonists of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor for activating metabolic enzymes such as those in the cytochrome P450 (CYP) superfamily, the expression of AhR was detected by real-time RT-PCR in the kidneys of wild-type and PCK rats. At 4-weeks of age with early stage PKD, the expression levels of AhR were significantly lower in PKD compared with wild-type kidneys. Intriguingly, its expression level in 20-week-old PCK kidneys with progressive stage PKD was higher compared with wild-type kidneys. Since cysts are derived from medulla in PCK kidneys, the distribution of AhR was observed by immunofluorescence mainly in renal medullary specimens. AhR is known to translocate into the nucleus upon ligand binding, and in normal kidneys at 4 weeks of age, AhR was localized to the nucleus in tubular epithelia, but not at 20 weeks of age in normal kidneys. However, in PCK kidneys at 20 weeks of age, AhR was localized in the nucleus in normal tubular epithelia and in developed cysts, while in dilated renal tubules (which develop into cysts), AhR translocated to the nucleus in PCK kidneys both at 4 and 20 weeks of age. Indolamine-2,3-dioxygenase 2 (IDO2) is an enzyme which catalyzes the conversion from tryptophan to kynurenine/kynurenic acid. In epithelial cells of dilated tubules in PCK kidneys, IDO2 was localized in the cytoplasm, suggesting that IDO2 may have a role in activating the AhR signaling pathway in cystogenesis. The current findings suggest that the progression of PKD may relate to the increased activation of tryptophan metabolism in combination with altered expression and distribution of AhR. Regulation of the kynurenine/kynurenic acid and AhR signaling pathway may have therapeutic potential against disease progression in PKD.

Malpighamoeba infection compromises fluid secretion and P-glycoprotein detoxification in Malpighian tubules

Malpighian tubules, analogous to vertebrate nephrons, play a key role in insect osmoregulation and detoxification. Tubules can become infected with a protozoan, Malpighamoeba, which damages their epithelial cells, potentially compromising their function. Here we used a modified Ramsay assay to quantify the impact of Malpighamoeba infection on fluid secretion and P-glycoprotein-dependent detoxification by desert locust Malpighian tubules. Infected tubules have a greater surface area and a higher fluid secretion rate than uninfected tubules. Infection also impairs P-glycoprotein-dependent detoxification by reducing the net rhodamine extrusion per surface area. However, due to the increased surface area and fluid secretion rate, infected tubules have similar total net extrusion per tubule to uninfected tubules. Increased fluid secretion rate of infected tubules likely exposes locusts to greater water stress and increased energy costs. Coupled with reduced efficiency of P-glycoprotein detoxification per surface area, Malpighamoeba infection is likely to reduce insect survival in natural environments.

TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis.

Rationale: Enhancing non–CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca2+-activated Cl− channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.

Stem cells from exfoliated deciduous teeth alleviate hyposalivation caused by Sjögren syndrome.

To evaluate the effect of stem cells from exfoliated deciduous teeth on the hyposalivation caused by Sjögren syndrome (SS) and investigate the mechanism. Stem cells were injected into the tail veins of non-obese diabetic mice, the animal model of SS. The saliva flow was measured after pilocarpine intraperitoneal injection. Apoptosis and autophagy were evaluated by TUNEL and Western blot. Lymphocyte proportions were detected by flow cytometer. Fluid secretion was decreased in 21-week-old mice. Stem cell treatment increased fluid secretion, alleviated inflammation in the submandibular glands and reduced inflammatory cytokine levels in the serum, submandibular glands and saliva. Stem cells decreased the apoptotic cell number and the expressions of ATG5 and Beclin-1 in the submandibular glands. Stem cells have no effect on other organs. Furthermore, the infused stem cells migrated to the spleen and liver, not the submandibular gland. Stem cells directed T cells towards Treg cells and suppressed Th1 and Tfh cells in spleen lymphocytes. Stem cells from exfoliated deciduous teeth alleviate the hyposalivation caused by SS via decreasing the inflammatory cytokines, regulating the inflammatory microenvironment and decreasing the apoptosis and autophagy. The stem cells regulated in T-cell differentiation are involved in the immunomodulatory effects.

Pharmacological and clinical profile of linaclotide (Linzess®), a novel therapeutic agent for irritable bowel syndrome with constipation and chronic constipation

Linaclotide (Linzess® tablets 0.25 mg) is a guanylate cyclase-C (GC-C) agonist with high selectivity and binding affinity to GC-C. In Japan, linaclotide was approved for 〝irritable bowel syndrome with constipation (IBS-C)〟 in December 2016 and 〝chronic constipation (CC) (excluding constipation due to organic disease)〟 in August 2018. Non-clinical studies demonstrated that linaclotide binding to GC-C increases intracellular cyclic guanosine monophosphate (cGMP), resulting in increased fluid secretion and gastrointestinal transit. In rats with colonic hyperalgesia, but not in normal rats, linaclotide suppressed the visceral nociceptive response, mediated by increased submucosal cGMP. In clinical studies in Japan, improvements were observed in the responder rates for global assessment of IBS symptom relief, complete spontaneous bowel movements in patients with IBS-C, and the frequency of spontaneous bowel movement in patients with CC, which were maintained during long-term treatment. Additionally, abdominal bloating, which has been associated with lower quality of life (QOL) and lower satisfaction with other approved therapies, and IBS QOL were improved throughout treatment with linaclotide. Diarrhea, a consequence of linaclotide's mechanism of action, was observed during the clinical studies, but was generally controllable by decreasing the linaclotide dose. No drug resistance was observed during the clinical studies, unlike some other approved agents. These results of non-clinical and clinical studies demonstrate that linaclotide can improve constipation, various abdominal symptoms, and QOL with a favorable safety profile in patients with IBS-C and CC.

Human evolutionary loss of epithelial Neu5Gc expression and species-specific susceptibility to cholera.

While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.

Differences in cell proliferation in the cpk and Pkd1 mouse models of polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common potentially fatal single gene disorders, resulting from mutations in either the Pkd1 or Pkd2 gene. Renal pathologies found in ADPKD include cysts resulting from increased fluid secretion, cell proliferation, and apoptosis, with an altered differentiation of the epithelial cells lining the cysts. In humans, ADPKD is thought to result from an inherited mutation in the PKD1 gene, followed by a somatic “second hit” mutation in the normal allele. The mechanism of this second hit is not understood. We analyzed the genomic sequence of human PKD1 and found extensive tandem repeats of guanine capable of supporting guanine quadruplex (G4) structures. G4 DNA is four‐stranded, and it is known to functionally contribute to recombination and mutagenesis. By comparison, mice do not carry extensive G4 repeats in the Pkd1 gene, thus targeted deletion of both Pkd1 alleles are required to generate cysts. However, homozygous Pkd1 null mice are embryonically lethal. Therefore, we have generated mice carrying a kidney specific deletion of the Pkd1 gene, called Pkd1CD. These mice develop rapid cystic disease and die by 14 days of age. Another commonly studied mouse model of PKD is a mutation in the cystin gene (cpk mice), which is a mouse model of ARPKD. These mice have a complete loss of the cystin gene, however, they survive until about three weeks of age. To begin to determine the differences in disease progression between the Pkd1CD mice and the cpk mice, we evaluated cell proliferation in these two models of PKD. While kidneys isolated from Pkd1CD mice showed high levels of cell proliferation in the cyst lining cells, similarly cystic kidneys from cpk mice showed very little cell proliferation in the cyst lining cells. Moreover, we observed differences between PCNA and Ki67 labeling as markers for cell proliferation in both mouse models. Because these mouse models differ significantly from human ADPKD, future studies will include replacing portions of the mouse PKD1 gene with the human PKD1 gene that includes the G4 repeats.Support or Funding InformationNIH DK100972This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

You Had Me at CRISPR: Exploring Gene Knockout Techniques in an Undergraduate Laboratory Course

A research project‐based Cell Biology laboratory serves as a capstone course for undergraduate students in the Biochemistry/Molecular Biology program at Benedictine university. This year, the students worked on a project to knockout a gene to study its function. Previous studies have implicated a role for CFTR in bile acid‐induced diarrhea. The gene‐editing technique, CRISPR (clustered regularly interspaced short palindromic repeats), was used to knockout the cystic fibrosis transmembrane conductance regulation (CFTR) gene to confirm its role in bile aciddiarrhea. In mammalian intestines, excess luminal bile acids affect the growth of colonic epithelial cells and increase fluid secretion resulting in fluid loss.The students were challenged to generate a cell line model ± CFTR. A common intestinal cell model, the T84 cell line derived from human colon carcinoma, was used to study CFTR function. However, these cells do not lend easily to genetic manipulation. Thus, the goal of the students in this course was to use the novel, efficient CRISPR/Cas9 technology to knockout CFTR function in T84 cells and compare the transfection efficiencies of two methods i) CRISPR/Cas9 system, utilizing an sgRNA expressing plasmid and ii) sgRNA in vitro transcription system, to deliver a complete transcribed molecule without a plasmid intermediary.Four groups of students having prior basic molecular biology technique experience learned cell culture techniques, read primary literature, including CRISPR and designed the primers for the constructs necessary for these studies. The CRISPR/Cas9 or the sgRNA construct with a Cas9 plasmid were introduced into T84 cells and the transcription efficiencies of both techniques were evaluated using real time PCR, Western blotting and T7 endonuclease assay. Following successful knockdown of CFTR, WT and CFTR−/− cells, grown in 12‐well plates, were incubated with primary and secondary bile acids and Cl− secretion was measured using MQAE fluorescence.Cell lines provide an excellent reductionist model to study CFTR expression and to introduce students to cell and molecular biology techniques. Students were excited to learn and successfully used innovative techniques to answer research questions in a classroom setting. Presented as a 3‐hour laboratory twice a week, most of the students succeeded in their projected goal to knockout CFTR in one semester, albeit some with low efficiencies. The course was conducted as an active learning classroom, where group activity and participation were essential. Beyond developing scientific expertise, the course further fostered scientific thinking, critical analysis and problem‐solving skills. For example, initial transfection efficiencies of only 3 – 5% were increased to 30–40% by the collaborative trouble shooting efforts of students. Scientific writing skills were honed using revise‐rewrite‐resubmit process whereby students produced multiple faculty‐reviewed and peer‐reviewed drafts of abstracts and publication style reports. They also got the experience of designing poster/slideshow and giving multimedia presentations at research symposia highlighting their findings at local and regional meetings, with documented positive responses given by visiting faculty from other local institutions. Further, these studies helped identify a simple, state of the art method to mediate genome editing in T84 cells, a model that typically has low transfection rates.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Institutional Funds, Ben. U. to JS and MPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Use of Fluorescein Isothiocyanate-Inulin as a Marker for Intestinal Ischemic Injury

Intestinal ischemia is observed in conditions such as mesenteric ischemia, or during traumatic events such as intestinal transplantation. Intestinal ischemia leads to pathophysiologic disruptions that present as increased fluid secretion into the intestinal lumen. We propose a novel method to detect real-time ischemic injury that is used in an invitro model applicable to intestinal transplantation. Small intestine segments from rats were procured. The segments were attached to customized perfusion chambers. Both intestines were perfused on the vascular side with a Ringer buffer solution. The experimental buffer solution was bubbled with 100% nitrogen to mimic ischemia. Both lumens were perfused with 3 mL HEPES-Ringer solution containing 50 μM fluorescein isothiocyanate (FITC)-inulin. Intraluminal samples were collected at 15-minute intervals to measure FITC-inulin concentration using a nanofluorospectrophotometer. Intestinal tissue samples were processed and evaluated by a blinded pathologist using the Park/Chiu scoring system for grading intestinal ischemia. Samples collected from the ischemic intestine showed a significant decrease in FITC-inulin fluorescence compared with the control intestine, indicating enhanced fluid secretion. Histopathologic samples from the experimental arm exhibited higher scores of ischemic injury in comparison with the control arm, confirming the FITC-inulin as a correlation to ischemia. Fluorescein isothiocyanate-inulin can be used as a real-time volume marker to monitor the ischemic state of intestinal tissue. A positive correlation between the degree of fluid shift and presence of ischemic injury. The changes in fluorescence signal provide a potential selective method to measure real-time fluid changes inside an intestinal graft to evaluate viability.

Secretory diarrhoea: mechanisms and emerging therapies.

Diarrhoeal disease remains a major health burden worldwide. Secretory diarrhoeas are caused by certain bacterial and viral infections, inflammatory processes, drugs and genetic disorders. Fluid secretion across the intestinal epithelium in secretory diarrhoeas involves multiple ion and solute transporters, as well as activation of cyclic nucleotide and Ca(2+) signalling pathways. In many secretory diarrhoeas, activation of Cl(-) channels in the apical membrane of enterocytes, including the cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, increases fluid secretion, while inhibition of Na(+) transport reduces fluid absorption. Current treatment of diarrhoea includes replacement of fluid and electrolyte losses using oral rehydration solutions, and drugs targeting intestinal motility or fluid secretion. Therapeutics in the development pipeline target intestinal ion channels and transporters, regulatory proteins and cell surface receptors. This Review describes pathogenic mechanisms of secretory diarrhoea, current and emerging therapeutics, and the challenges in developing antidiarrhoeal therapeutics.

Identification, functional characterization, and pharmacological profile of a serotonin type-2b receptor in the medically important insect, Rhodnius prolixus.

In the Chagas disease vector, Rhodnius prolixus, two diuretic hormones act synergistically to dramatically increase fluid secretion by the Malpighian tubules (MTs) during the rapid diuresis that is initiated upon engorgement of vertebrate blood. One of these diuretic hormones is the biogenic amine, serotonin (5-hydroxytryptamine, 5-HT), which controls a variety of additional activities including cuticle plasticization, salivary gland secretion, anterior midgut absorption, cardioacceleratory activity, and myotropic activities on a number of visceral tissues. To better understand the regulatory mechanisms linked to these various physiological actions of serotonin, we have isolated and characterized a serotonin type 2b receptor in R. prolixus, Rhopr5HTR2b, which shares sequence similarity to the vertebrate serotonin type 2 receptors. Rhopr5HTR2b transcript is enriched in well-recognized physiological targets of serotonin, including the MTs, salivary glands and dorsal vessel (i.e., insect heart). Notably, Rhopr5HTR2b was not enriched in the anterior midgut where serotonin stimulates absorption and elicits myotropic control. Using a heterologous functional receptor assay, we examined Rhopr5HTR2b activation characteristics and its sensitivity to potential agonists, antagonists, and other biogenic amines. Rhopr5HTR2b is dose-dependently activated by serotonin with an EC50 in the nanomolar range. Rhopr5HTR2b is sensitive to alpha-methyl serotonin and is inhibited by a variety of serotonin receptor antagonists, including propranolol, spiperone, ketanserin, mianserin, and cyproheptadine. In contrast, the cardioacceleratory activity of serotonin revealed a unique pharmacological profile, with no significant response induced by alpha-methyl serotonin and insensitivity to ketanserin and mianserin. This distinct agonist/antagonist profile indicates that a separate serotonin receptor type may mediate cardiomodulatory effects controlled by serotonin in R. prolixus.

Vasopressin and disruption of calcium signalling in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disease and is responsible for 5-10% of cases of end-stage renal disease worldwide. ADPKD is characterized by the relentless development and growth of cysts, which cause progressive kidney enlargement associated with hypertension, pain, reduced quality of life and eventual kidney failure. Mutations in the PKD1 or PKD2 genes, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause ADPKD. However, neither the functions of these proteins nor the molecular mechanisms of ADPKD pathogenesis are well understood. Here, we review the literature that examines how reduced levels of functional PC1 or PC2 at the primary cilia and/or the endoplasmic reticulum directly disrupts intracellular calcium signalling and indirectly disrupts calcium-regulated cAMP and purinergic signalling. We propose a hypothetical model in which dysregulated metabolism of cAMP and purinergic signalling increases the sensitivity of principal cells in collecting ducts and of tubular epithelial cells in the distal nephron to the constant tonic action of vasopressin. The resulting magnified response to vasopressin further enhances the disruption of calcium signalling that is initiated by mutations in PC1 or PC2, and activates downstream signalling pathways that cause impaired tubulogenesis, increased cell proliferation, increased fluid secretion and interstitial inflammation.

Simultaneous exposure to Escherichia coli heat-labile and heat-stable enterotoxins increases fluid secretion and alters cyclic nucleotide and cytokine production by intestinal epithelial cells.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease and death, especially in children in developing countries. ETEC causes disease by colonizing the small intestine and producing heat-labile toxin (LT), heat-stable toxin (ST), or both LT and ST (LT+ST). The majority of ETEC strains produce both ST and LT. Despite the prevalence of LT+ST-producing organisms, few studies have examined the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins. In the current report, we demonstrate that when LT and ST are both present, they increase water movement into the intestinal lumen over and above the levels observed with either toxin alone. As expected, cultured intestinal epithelial cells increased their expression of intracellular cyclic GMP (cGMP) when treated with ST and their expression of intracellular cyclic AMP (cAMP) when treated with LT. When both toxins were present, cGMP levels but not cAMP levels were synergistically elevated compared with the levels of expression caused by the corresponding single-toxin treatment. Our data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells in response to LT are significantly reduced in animals exposed to both enterotoxins. These findings suggest that there may be complex differences between the epithelial cell intoxication and, potentially, secretory outcomes induced by ETEC strains expressing LT+ST compared with strains that express LT or ST only. Our results also reveal a novel mechanism wherein ST production may reduce the hosts' ability to mount an effective innate or adaptive immune response to infecting organisms.

Testosterone decreases fluid and chloride secretions in the uterus of adult female rats via down-regulating cystic fibrosis transmembrane regulator (CFTR) expression and functional activity

ObjectivesEstrogen is known to stimulate uterine fluid and Cl− secretion via CFTR. This study investigated testosterone effect on these changes in a rat model. MethodsOvariectomized adult female rats received estrogen for five days or estrogen for three days followed by two days peanut oil or testosterone either alone or in the presence of flutamide or finasteride. At the end of treatment, uteri were perfused with perfusate containing CFTRinh-172. The rate of fluid and Cl− secretion were determined. Dose-dependent effect of testosterone and effect of forskolin on fluid secretion rate were measured. Animals were sacrificed and uteri were removed for CFTR protein and mRNA expression analyses, histology and cAMP measurement. Morphology of uterus, levels of expression of CFTR protein and mRNA and distribution of CFTR protein were observed. ResultsEstrogen causes increase while testosterone causes decrease in uterine fluid and Cl− secretions. The effects of estrogen but not testosterone were antagonized by CFTRinh-172. Luminal fluid volume and apical expression of CFTR in the luminal epithelia were highest under estrogen and lowest under testosterone influences. Similar changes were observed in CFTR protein and mRNA expressions. Uterine cAMP level was highest under estrogen and lowest under testosterone influence. Forskolin increases fluid secretion rate in estrogen but not in testosterone-treated animals. Testosterone effects were dose-dependent and were antagonized by flutamide however, not finasteride. ConclusionsTestosterone inhibition of estrogen-induced uterine fluid and Cl− secretion occurs via inhibition of CFTR expression and functional activities. These changes could explain the adverse effects of testosterone on fertility.

Editorial Comment on: Andrzej Smereczyński, Teresa Starzyńska, Katarzyna Kołaczyk and Józef Kładny Role of sonography in assessing complications after laparoscopic cholecystectomy.

Laparoscopic cholecystectomy (LC) was introduced to the arsenal of surgical techniques in 1987 and has become the gold standard in treating cholecystolithiasis. In spite of undoubted benefits for patients, it is not a complication-free procedure. The type of complications after a classical, i.e. “open” surgery and LC do not differ in a significant way. The difference lies in their prevalence. One of the most common complications is a fluid collection in the gallbladder bed under the liver. To prevent it from developing, a drain is usually placed under the liver. For many years, it was believed that such a practice is correct, but recent reports indicate that leaving the drain after a planned laparoscopic cholecystectomy may do more damage rather than be beneficial. Moreover, it may give a false impression of safety since we are convinced that we remain in control over the situation in case of, for instance, a hemorrhage(1). In fact, when the postoperative bleeding to the peritoneal cavity is intense, a thin Redon drain, which usually remains inserted after the procedure, may become occluded by a thrombus. Decisions are made based on the patient's clinical condition or imaging examinations (including sonography) which can reveal not only a fluid collection under the liver, but also free fluid in the peritoneal cavity. The peritoneum secretes considerable amounts of peritoneal fluid daily. It becomes absorbed completely mainly via the lymphatic channels in the diaphragmatic region. Therefore, detection of a collection of serous fluid should not be alarming. It will absorb with no consequences for the patient. It has been observed that leaving a drain in the gallbladder bed causes increased fluid secretion. Moreover, Fraser et al. claim that such a practice may provoke bile leakage form slight bile ducts in the gallbladder bed(2). If it is a suction drain the risk of bile leakage is significant. Thiebe and Eggert observed that leaving the drain caused fluid to accumulate in the gallbladder bed in 44% of cases compared to 4.1% in the group without the drain(3). Their results were confirmed in ultrasound examinations which revealed that the fluid collection that developed within 24 hours after the surgery appeared considerably more frequently in the group with the drain. When, apart from ultrasound findings, patients also manifest symptoms of peritoneal irritation, the clinical picture becomes slightly different. In such a situation, we deal with bile leakage, either from damaged bile ducts of from the duct of Luschka. This can be managed by an endoscopic intervention or a US-guided puncture and drainage of the fluid collection. If the presence of fluid is concomitant with elevated temperature and general symptoms of inflammation, an abscess may be suspected to have formed under the liver. In such a situation, a surgical intervention is also necessary. To conclude, benefits of leaving a drain in the gallbladder bed after uncomplicated cholecystectomy are not significant. Numerous studies reveal that removing the drain results in fewer pain complaints, better comfort of patients, lower rate of complications and faster discharge from hospital. Furthermore, pain complaints associated with the presence of the drain restrict chest movement at the right side and result in more frequent respiratory infections. The role of sonography in the assessment of fluid collections in the peritoneal cavity following laparoscopic cholecystectomy is not overrated. An ultrasound examination is simple, repeatable, safe and can be a remarkable diagnostic and therapeutic tool in experienced hands. However, ultrasound findings must not be interpreted separately from the clinical picture. Thanks to a skillful combination of these elements, it is possible to avoid numerous unneeded instrumental actions which do not always give an expected therapeutic effect. The remarks and tips presented by Smereczynski et al.(4) regarding the usage of various diagnostic tools in the post-operative period, particularly in diagnosing complications, are of considerable practical value and are useful for every surgeon irrespective of his or her experience.

Effect of the bile acids unconjugated and conjugated, chenodeoxycholic acid and deoxycholic acid, on epithelial tight junction barrier function in a human colon carcinoma cell line, T84 (1113.3)

The human colon is exposed to cBA, and ucBA and excess BAs can increase fluid secretion and perturb the epithelial barrier. We showed in T84 cells (AJP 305: C447, ’13) that: a) BAs preferentially stimulate Cl‐ secretion from the basolateral surface (BLM); b) Overnight exposure (O/N) to proinflammatory cytokine (PiC), decreases transepithelial electrical resistance (TER) and then, luminal (AM) BA can stimulate Cl‐ secretion. We hypothesize that high AM [BAs] directly decrease TER, and this is enhanced by PiC. Confluent T84 cells (TER >1000 Ohm.cm2) were treated with 5‐500µM BAs for 0.5‐24h + PiCs (TNFα+IL‐1ß+IFNγ). Cell viability and toxicity were assessed by Accuri flow cytometry and lactate dehydrogenase respectively and barrier function by TER and dextran fluxes. Viability and cytotoxicity were dose and time dependent. UcBAs (500μM, O/N) had higher cytotoxicity (CDCA:28.6%; DCA:27.8%) than cBAs (TCDC:15.5%; TDC:13.3%). Physiological doses (5‐50μM) were less toxic (8‐10%). O/N PiC decreased TER by ~50% and 0.5mM CDCA (20’) decreased this further by 20%. AM, not BLM CDCA (0.5mM), increased (~2‐fold) 10‐kDa blue‐dextran AM to BLM flux over time (8h); PiC increased this further (+2‐fold). In summary, AM BAs alter epithelial barrier integrity allowing luminal BAs to access the BLM and this is enhanced by PiC. This will provide the basis for future studies on the role of BA‐induced diarrhea in inflammatory diseases.Grant Funding Source: Supported by COS, Benedictine U.

Review article: linaclotide for the management of irritable bowel syndrome with constipation

BackgroundIrritable bowel syndrome with constipation (IBS-C) represents a significant burden to patients and healthcare systems due to its prevalence and lack of successful symptomatic resolution with established treatment options. Linaclotide 290 μg has recently been approved by the European Medicines Agency (EMA) for moderate-to-severe IBS-C and by the US Food and Drug Administration for IBS-C (290 μg dose) and for chronic constipation (145 μg dose).AimTo summarise data leading to the approval of linaclotide for IBS-C, with focus on EMA-pre-specified outcome measures.MethodsLiterature search of a peer-review database (PubMed) and review of congress abstracts on linaclotide preclinical and clinical trial data in IBS-C.ResultsPreclinical studies suggest that the guanylate cyclase C agonist (GCCA) linaclotide acts through elevation of cyclic guanosine monophosphate (cGMP) levels, leading to accelerated gastrointestinal (GI) transit through increased fluid secretion and reduced visceral hypersensitivity. Clinical trial data demonstrate that linaclotide improves abdominal symptoms (pain, bloating) and bowel symptoms (constipation) compared with placebo in patients with IBS-C. The most frequent side effect, diarrhoea, results from the therapeutic action of linaclotide. Linaclotide acts locally in the GI tract with minimal systemic exposure, resulting in low oral bioavailability and thus a low risk of relevant systemic adverse effects.ConclusionLinaclotide, a first-in-class GCCA, is a promising new drug with a novel, dual mechanism of action that, unlike more well-established agents, can relieve the abdominal pain, bloating and constipation associated with IBS-C and has a low propensity for systemic side effects.