Intestinal mucosal perfusion and integrity are maintained in hypotensive brain dead mice.

Brain death (BD) leads to complex hemodynamic and inflammatory alterations which may compromise organ perfusion and induce morphologic and functional damage in various organs. The intestine is particularly sensitive to hypoperfusion and donor hypotension usually precludes intestinal donation. Previous studies reported inflammatory intestinal changes following BD but information on mucosal integrity and perfusion are lacking. BD was induced in mice by inflating an epidural balloon catheter. Controls underwent only anesthesia and tracheostomy. Intestinal perfusion was assessed using laser Doppler flowmetry (LDF). Intestinal injury was assessed after 2h of BD by the Chiu-Park score and morphometry. Intestinal tight junction (TJ) proteins (claudin-1, claudin-3, occludin, tricellulin) as well as inflammatory activation (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin-6) were also analysed and compared with a sham group. Although blood pressure decreased in BD mice, intestinal perfusion remained similar between BD and sham mice. Histologically, mucosal injury was absent/minimal and TJs appeared well maintained in both groups. BD may trigger intrinsic, autoregulatory mechanisms to preserve microvascular tissue perfusion and mucosal integrity in spite of mild hypotension.

Intestinal Ketogenesis and Permeability.

Consumption of a high-fat diet (HFD) has been suggested as a contributing factor behind increased intestinal permeability in obesity, leading to increased plasma levels of microbial endotoxins and, thereby, increased systemic inflammation. We and others have shown that HFD can induce jejunal expression of the ketogenic rate-limiting enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS). HMGCS is activated via the free fatty acid binding nuclear receptor PPAR-α, and it is a key enzyme in ketone body synthesis that was earlier believed to be expressed exclusively in the liver. The function of intestinal ketogenesis is unknown but has been described in suckling rats and mice pups, possibly in order to allow large molecules, such as immunoglobulins, to pass over the intestinal barrier. Therefore, we hypothesized that ketone bodies could regulate intestinal barrier function, e.g., via regulation of tight junction proteins. The primary aim was to compare the effects of HFD that can induce intestinal ketogenesis to an equicaloric carbohydrate diet on inflammatory responses, nutrition sensing, and intestinal permeability in human jejunal mucosa. Fifteen healthy volunteers receiving a 2-week HFD diet compared to a high-carbohydrate diet were compared. Blood samples and mixed meal tests were performed at the end of each dietary period to examine inflammation markers and postprandial endotoxemia. Jejunal biopsies were assessed for protein expression using Western blotting, immunohistochemistry, and morphometric characteristics of tight junctions by electron microscopy. Functional analyses of permeability and ketogenesis were performed in Caco-2 cells, mice, and human enteroids. Ussing chambers were used to analyze permeability. CRP and ALP values were within normal ranges and postprandial endotoxemia levels were low and did not differ between the two diets. The PPARα receptor was ketone body-dependently reduced after HFD. None of the tight junction proteins studied, nor the basal electrical parameters, were different between the two diets. However, the ketone body inhibitor hymeglusin increased resistance in mucosal biopsies. In addition, the tight junction protein claudin-3 was increased by ketone inhibition in human enteroids. The ketone body ß-Hydroxybutyrate (ßHB) did not, however, change the mucosal transition of the large-size molecular FD4-probe or LPS in Caco-2 and mouse experiments. We found that PPARα expression was inhibited by the ketone body ßHB. As PPARα regulates HMGCS expression, the ketone bodies thus exert negative feedback signaling on their own production. Furthermore, ketone bodies were involved in the regulation of permeability on intestinal mucosal cells in vitro and ex vivo. We were not, however, able to reproduce these effects on intestinal permeability in vivo in humans when comparing two weeks of high-fat with high-carbohydrate diet in healthy volunteers. Further, neither the expression of inflammation markers nor the aggregate tight junction proteins were changed. Thus, it seems that not only HFD but also other factors are needed to permit increased intestinal permeability in vivo. This indicates that the healthy gut can adapt to extremes of macro-nutrients and increased levels of intestinally produced ketone bodies, at least during a shorter dietary challenge.

Mucosal Recovery after Intestinal Transplantation in the Rat: A Sequential Histological and Molecular Assessment.

INTRODUCTION: Intestinal cold ischemia and subsequent reperfusion during transplantation result in various degrees of mucosal injury ranging from mild edema to extensive mucosal loss. Mucosal barrier impairment favors bacterial translocation and fluid loss and raises nutritional challenges. The injured intestine also releases proinflammatory mediators and upregulates various epitopes toward an inflammatory phenotype. We studied the process of mucosal injury and repair during the early period after intestinal transplantation from a histological and molecular standpoint. MATERIALS AND METHODS: Adult Sprague-Dawley rats were used as donors and recipients. Donor intestines were perfused and stored in saline for 3 h, then transplanted heterotopically using microvascular anastomoses. Intestinal graft segments were obtained after 20 min, 6 h, 12 h, and 24 h after reperfusion. Histology studies (goblet cell count, morphometry), immunofluorescence, and western blot for several tight junction proteins, apoptosis, and inflammation-related proteins were performed. RESULTS: Cold storage led to extensive epithelial detachment, whereas reperfusion resulted in extensive villus loss (about 60% of the initial length), and goblet cell numbers were drastically reduced. Over the first 24 h, gradual morphologic and molecular recovery was noted, although several molecular alterations persisted (increased apoptosis and inflammation, altered expression of several tight junctions). CONCLUSIONS: The current data suggest that a near-complete morphologic recovery from a moderate mucosal injury occurs within the first 24 h after intestinal transplantation. However, several molecular alterations persist and need to be considered when designing intestinal transplant experiments and choosing sampling and endpoints.

Luminal polyethylene glycol solution delays the onset of preservation injury in the human intestine.

The organ damage incurred during the cold storage (CS) of intestinal grafts has short and long-term consequences. Animal studies suggest that additional luminal preservation (LP) with polyethylene glycol (PEG) may alleviate this damage. This study aims to validate these findings using human intestines. Ileal segments, perfused intravascularly with IGL-1 solution, were procured from 32 multiorgan donors and divided into two parts: one containing a PEG 3350-based solution introduced luminally (LP group) and another one without luminal treatment (control). Sampling was performed after 4 h, 8 h, 14 h, and 24 h of CS. Histology was assessed using the Chiu/Park score. Tight junctions (TJ), several inflammatory markers, and transcription factors were examined by immunofluorescence, ddPCR, and western blot. Tissue water content (edema) was also measured. Apoptotic activity was assessed with caspase -2, -3, and -9 assays. LP significantly lowered mucosal injury at all time points. Redistribution of TJ proteins occurred earlier and more severely in the control group. After 24 h of CS, LP intestines showed an emerging unfolding protein response. Increased caspase-3 and -9 activity was found in the control group. The current results indicate that luminal PEG is safe and effective in reducing damage to the intestinal epithelium during CS.

Suppression of enteroendocrine cell glucagon-like peptide (GLP)-1 release by fat-induced small intestinal ketogenesis: a mechanism targeted by Roux-en-Y gastric bypass surgery but not by preoperative very-low-calorie diet.

OBJECTIVE: Food intake normally stimulates release of satiety and insulin-stimulating intestinal hormones, such as glucagon-like peptide (GLP)-1. This response is blunted in obese insulin resistant subjects, but is rapidly restored following Roux-en-Y gastric bypass (RYGB) surgery. We hypothesised this to be a result of the metabolic changes taking place in the small intestinal mucosa following the anatomical rearrangement after RYGB surgery, and aimed at identifying such mechanisms. DESIGN: Jejunal mucosa biopsies from patients undergoing RYGB surgery were retrieved before and after very-low calorie diet, at time of surgery and 6 months postoperatively. Samples were analysed by global protein expression analysis and Western blotting. Biological functionality of these findings was explored in mice and enteroendocrine cells (EECs) primary mouse jejunal cell cultures. RESULTS: The most prominent change found after RYGB was decreased jejunal expression of the rate-limiting ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGCS), corroborated by decreased ketone body levels. In mice, prolonged high-fat feeding induced the expression of mHMGCS and functional ketogenesis in jejunum. The effect of ketone bodies on gut peptide secretion in EECs showed a ∼40% inhibition of GLP-1 release compared with baseline. CONCLUSION: Intestinal ketogenesis is induced by high-fat diet and inhibited by RYGB surgery. In cell culture, ketone bodies inhibited GLP-1 release from EECs. Thus, we suggest that this may be a mechanism by which RYGB can remove the inhibitory effect of ketone bodies on EECs, thereby restituting the responsiveness of EECs resulting in increased meal-stimulated levels of GLP-1 after surgery.

GM1 ganglioside-independent intoxication by Cholera toxin.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.

The human duodenal mucosa harbors all components for a local renin angiotensin system.

The renin-angiotensin system (RAS) is present in the gastrointestinal (GI) tract but remains to be fully characterized, particularly in man. The duodenum plays a role in both the upper and lower GI regulation, as well as in distant organs. The present study investigates the presence and functional potential of RAS in the human duodenal mucosa of healthy individuals. Endoscopically acquired mucosal biopsies from healthy volunteers were examined using western blot, immunohistochemistry, and ELISA. Functionality was examined by using Ussing chambers and recording duodenal transmucosal potential difference (PD) and motility in vivo Angiotensinogen, Angiotensin II (AngII) and its receptors (AT1R, AT2R) as well as to the RAS associated enzymes renin, ACE, and neprylisin were detected in all samples of duodenal mucosa. Migrating motility complex induced elevations of transmucosal PD were significantly larger after per-oral administration of the AT1R receptor antagonist candesartan. Fasting duodenal motility per se was not influenced by candesartan. The epithelial current produced by duodenal mucosae mounted in Ussing chambers increased significantly after addition of AngII to specimens where the AT1R was blocked using losartan. The epithelial current also increased after addition of the AT2R-selective agonist C21. Immunostaining and pharmacological data demonstrate the presence of a local RAS in the human duodenal mucosa with capacity to influence epithelial ion transport by way of particulary the AT2R.

Intestinal Preservation Injury: A Comparison Between Rat, Porcine and Human Intestines.

Advanced preservation injury (PI) after intestinal transplantation has deleterious short- and long-term effects and constitutes a major research topic. Logistics and costs favor rodent studies, whereas clinical translation mandates studies in larger animals or using human material. Despite diverging reports, no direct comparison between the development of intestinal PI in rats, pigs, and humans is available. We compared the development of PI in rat, porcine, and human intestines. Intestinal procurement and cold storage (CS) using histidine-tryptophan-ketoglutarate solution was performed in rats, pigs, and humans. Tissue samples were obtained after 8, 14, and 24 h of CS), and PI was assessed morphologically and at the molecular level (cleaved caspase-3, zonula occludens, claudin-3 and 4, tricellulin, occludin, cytokeratin-8) using immunohistochemistry and Western blot. Intestinal PI developed slower in pigs compared to rats and humans. Tissue injury and apoptosis were significantly higher in rats. Tight junction proteins showed quantitative and qualitative changes differing between species. Significant interspecies differences exist between rats, pigs, and humans regarding intestinal PI progression at tissue and molecular levels. These differences should be taken into account both with regards to study design and the interpretation of findings when relating them to the clinical setting.

Luminal Polyethylene Glycol Alleviates Intestinal Preservation Injury Irrespective of Molecular Size.

Intestinal preservation injury (IPI) and the resulting mucosa injury raise several serious challenges early after intestinal transplantation. The current clinical approach using only vascular perfusion allows the shortest preservation period among the abdominal organs. The experimental addition of luminal polyethylene glycol (PEG) solutions has been repeatedly suggested to alleviate preservation injury, improve graft quality, and prolong the preservation time. We investigated whether the molecular mass of PEG in solution influences the development of intestinal preservation injury. Small intestines of Sprague-Dawley rats were perfused with University of Wisconsin solution. Group 1 underwent vascular perfusion only (clinical control), group 2 received additional luminal PEG3350 Da, group 3 received luminal PEG10000 Da, and group 4 received luminal PEG20000 Da (n = 8/group). Tissue samples were obtained after 4, 8, and 14 hours. We studied the tissue damage (Chiu/Park score, Goblet cells, apoptosis, tight junctions), activation of c-Jun NH2-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK), and we performed Ussing chamber assessments. Mucosal morphologic and electrophysiologic parameters were significantly improved in the groups receiving luminal PEG. There was significantly less apoptotic activity in groups 2, 3, and 4. Both MAPKs revealed an activation peak after 4 hours with group 3 showing lesser p38-MAPK activation. PEG 20 kDa interfered with protein immunodetection. The results indicate that luminal solutions of PEG of medium and large molecular mass significantly delay the onset and development of IPI, providing further evidence that luminal interventions may allow for longer cold storage intervals of intestinal grafts.

A sequential assessment of the preservation injury in porcine intestines.

BACKGROUND: Clinical and experimental evidence strongly suggest that ischemia-reperfusion injury after intestinal transplantation has deleterious short- and long-term effects and finding means to reduce ischemia-reperfusion injury is a major research area. The anatomical and physiological similarities between the human and porcine digestive tract favor its use as a preclinical model for translational research. Intriguingly, no systematic appraisal of the development of the intestinal preservation injury in pigs is available. MATERIALS AND METHODS: Intestinal procurement was performed in nine pigs using histidine-tryptophan-ketoglutarate solution as preservation fluid. Ileal biopsies were obtained after 8, 14, and 24 h of static cold storage (SCS), and the preservation injury was assessed morphologically (Chiu score) as well as on the molecular level. Tight junction (zonula occludens, claudin-3 and 4, tricellulin, and occludin) and adherens junctions (E-cadherin) proteins were studied using immunofluorescence and Western blot. RESULTS: Eight hours of SCS induced minimal mucosal changes (Chiu grade 1) that advanced to significant subepithelial edema (Chiu grade 3) after 24 h; progressive Goblet cell depletion was also noted. Apoptosis (studied by cleaved caspase-3 staining significantly increased after 24 h of SCS. Significant molecular changes with decreasing expression of zonula occludens, tricellulin, and occludin were evident already after 8 h of SCS and continuously worsened. Claudin-3 and Claudin-4 and E-cadherin expression remained relatively unaltered during SCS. CONCLUSIONS: Important molecular alterations precede histologic changes during SCS of the porcine intestine and may be used as more sensitive injury markers than histologic changes in intestinal ischemia and transplantation.

Support for involvement of the renin-angiotensin system in dysplastic Barrett's esophagus.

BACKGROUND AND AIM: Patients with dysplasia in Barrett's esophagus (BE) have a considerable risk of developing esophageal adenocarcinoma (EAC). The mucosal expression of the pro-inflammatory angiotensin II receptor type 1 (AT1R) is elevated in these patients, suggesting a role in carcinogenesis. The purpose of this study was to determine whether interference with the renin-angiotensin system (RAS) would influence downstream markers of carcinogenesis. METHODS: Endoscopic mucosal biopsies from BE patients with low-grade dysplasia (LGD) were sampled before and after a three-week period of RAS-interfering treatment. Thirty patients were randomly allocated to enalapril (ACE inhibitor, 5 mg od), candesartan (AT1R antagonist, 8 mg od), or no drug. The expression of 12 proteins known to be associated with RAS and carcinogenesis was assessed using western blot. RESULTS: We found altered expression of several proteins after enalapril treatment (decreased: NFκB, p = .043; NLRP3, p = .050; AMACR, p = .017; and caspase 3, p = .025; increased: p53, p = .050). Candesartan treatment was associated with increased iNOS expression (p = .033). No significant changes were seen in the no-drug group. CONCLUSION: Interference with angiotensin II formation was associated with altered expression of inflammation- and carcinogenesis-related proteins. The present results speak in favor of involvement of angiotensin II in BE dysplasia, but the role of AT1R should be investigated further.

The renin-angiotensin system in Barrett's esophagus.

OBJECTIVE: Barrett's esophagus (BE) is a risk factor for esophageal adenocarcinoma. In addition to its classical endocrine character known for hemodynamic regulation, the renin-angiotensin system (RAS) can be associated with inflammation, wound healing, and cancer. The aim of this study was to explore a potential expression of the RAS in BE, with or without the presence of dysplasia. MATERIAL AND METHODS: Biopsy material was prepared for western blotting and immunohistochemistry. Non-BE patients (controls) were compared with BE patients regarding RAS in the squamous epithelium. In the columnar BE mucosa, RAS expression was studied in patients with and without dysplasia. Key components of the 'classical' RAS were assessed: the angiotensin-converting enzyme (ACE) and the angiotensin II subtype 1 and 2 receptors (AT1R and AT2R). RESULTS: The presence of RAS factors was confirmed in the esophageal mucosa of both control and BE patients. ACE protein expression was 48% lower (p = 0.001) whereas AT1R was 45% higher (p = 0.039) in the squamous epithelium of BE patients compared to epithelia from non-BE controls. In the metaplastic intestinal-like epithelium, AT1R expression was 37% higher in BE patients with confirmed dysplasia than in patients without dysplasia (p = 0.009). Immunohistochemistry showed an altered distribution of RAS proteins in BE patients with dysplasia. CONCLUSIONS: The differential RAS expression observed may prove to be useful as a biomarker or a pharmaceutical target.

Luminal solutions protect mucosal barrier during extended preservation.

BACKGROUND: Mucosal barrier injury during intestinal preservation (IP) and transplantation favors life-threatening infections. Luminal delivery of solutions containing amino acids or polyethylene glycols (PEGs) may improve preservation results and reduce this injury. We tested if solutions containing glutamine and PEG influence the mucosal injury. MATERIALS AND METHODS: Rat intestines were perfused and stored in Viaspan-University of Wisconsin solution. Before IP, a PEG 3350 solution was introduced intraluminally alone (group 1) or supplemented with 40 mmol/L L-glutamine (group 2). Controls underwent vascular flush alone (group 3). Preservation injury was evaluated after 8, 14, and 24 h by histology and goblet cell count. Tight-junction proteins zonula occludens-1, claudin-3, claudin-4, and caveolin-1 were studied by immunofluorescence. Maltase and caspase-3 activity were also analyzed. RESULTS: Group 1 showed mild edema at 8 h and mucosal disruption by 24 h; these features were greatly improved in group 2 where continuous mucosa was found after 24 h of IP. Intestines in group 3 did worse at all time points with subepithelial edema (Park/Chiu grade 3) and marked goblet cell depletion; caspase-3 activity was lowest in group 2. Tight-junction proteins varied continuously during IP; zonula occludens-1 expression and colocalization with claudins decreased significantly in group 3 but not in other groups. Claudin-3 was distinctly localized in the membrane, but stained diffuse, cytoplasmic at later time-points. Claudin-4 changed to a cytoplasmic granular pattern. No caveolin-1 colocalization was observed. CONCLUSIONS: Luminal PEG and glutamine delay epithelial breakdown and preserve several important mucosal features during extended IP.

Angiotensin II exerts dual actions on sodium-glucose transporter 1-mediated transport in the human jejunal mucosa.

OBJECTIVES: Intestinal glucose absorption is mainly mediated via the sodium-glucose transporter 1 (SGLT1) at the apex of the enterocytes, whereas the glucose transporter 2 (GLUT2) provides a basolateral exit. It has been shown in rats that Angiotensin II (AngII), the principal mediator of renin-angiotensin system (RAS), inhibits jejunal SGLT1-mediated glucose absorption. The aim of the present study was to investigate if a similar mechanism exists also in the human jejunal mucosa. MATERIAL AND METHODS: Enteroscopy with mucosal biopsy sampling was performed in 28 healthy volunteers. Functional assessments were performed in Ussing chambers using a pharmacological approach. Western blotting and immunohistochemistry were used to assess the presence of the AngII type 1 (AT1R) and type 2 receptor (AT2R), as well as the glucose transporters SGLT1 and GLUT2. RESULTS: Exposure of the mucosa to 10 mM glucose elicited a ≈50% increase in the epithelium-generated current (Iep). This glucose-induced electrogenic response was sensitive to the competitive SGLT1 inhibitor phlorizin, but not to AngII when given alone. AngII combined with the AT2R blocker PD123319 markedly inhibited the response. AngII in combination with the AT1R antagonist losartan tended to increase the electrogenic response, whereas direct activation of AT2R using the agonist C21 significantly enhanced the mucosal response to glucose. The AT1R and AT2R as well as SGLT1 and GLUT2 were detected inside the human enterocytes. CONCLUSIONS: The pharmacological analysis indicated that activation of AT1R inhibits, whereas activation of AT2R enhances SGLT1-mediated glucose transport in the human jejunal mucosa.

Role of FFAR3 in ketone body regulated glucagon-like peptide 1 secretion.

Background: Roux-en-Y gastric bypass (RYGB) is an effective treatment for obesity, resulting in long-term weight loss and rapid remission of type 2 diabetes mellitus. Improved glucagon-like peptide 1 (GLP-1) levels is one factor that contributes to the positive effects. Prior to RYGB, GLP-1 response is blunted which can be attributed to intestinal ketogenesis. Intestinal produced ketone bodies inhibit GLP-1 secretion in enteroendocrine cells via an unidentified G-protein coupled receptors (GPCRs). A possible class of GPCRs through which ketone bodies may reach are the free fatty acid receptors (FFARs) located at the basolateral membrane of enteroendocrine cells. Aim: To evaluate FFAR3 expression in enteroendocrine cells of the small intestine under different circumstances, such as diet and bariatric surgery, as well as explore the link between ketone bodies and GLP-1 secretion. Materials and methods: FFAR3 and enteroendocrine cell expression was analyzed using Western blot and immunohistochemistry in biopsies from healthy volunteers, obese patients undergoing RYGB and mice. GLUTag cells were used to study GLP-1 secretion and FFAR3 signaling pathways. Results: The expression of FFAR3 is markedly influenced by diet, especially high fat diet, which increased FFAR3 protein expression. Lack of substrate such as free fatty acids in the alimentary limb after RYGB, downregulate FFAR3 expression. The number of enteroendocrine cells was affected by diet in the normal weight individuals but not in the subjects with obesity. In GLUTag cells, we show that the ketone bodies exert its blocking effect on GLP-1 secretion via the FFAR3, and the Gαi/o signaling pathway. Conclusion: Our findings that ketone bodies via FFAR3 inhibits GLP-1 secretion bring important insight into the pathophysiology of T2D. This highlights the role of FFAR3 as a possible target for future anti-diabetic drugs and treatments.

Esophageal barrier function and tight junction expression in healthy subjects and patients with gastroesophageal reflux disease: functionality of esophageal mucosa exposed to bile salt and trypsin in vitro.

BACKGROUND AND AIMS. Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. However, the influence of acid and/or bile acids on human esophageal epithelial barrier function and the tight junction (TJ) proteins has not been fully elucidated. The aim of the study is to investigate the esophageal barrier function and TJ expression in healthy subjects and patients with GERD. The functionality of esophageal mucosa exposed to bile salt deoxycholic acid (DCA) and trypsin has been studied in vitro. MATERIAL AND METHODS. Endoscopic biopsies from healthy controls and patients with GERD-related symptom with endoscopic erosive signs, as well as esophageal mucosa taken from patients undergoing esophagectomy were evaluated in Ussing chambers and by western blot and immunohistochemistry. RESULTS. The esophageal epithelium from GERD patients had lower electrical resistance and higher epithelial currents than controls. Claudin-1 and -4 were significantly decreased in GERD patients. The bile salt DCA in the low concentration of 1.5 mM and trypsin increased the resistance and claudin-1 expression, while the higher concentration of 2.5 mM DCA and trypsin decreased the resistance and the claudin-3, -4 and E-cadherin expressions. CONCLUSION. In addition to acidic reflux, duodenal reflux components, such as bile salts and trypsin, have the potential to disrupt the esophageal barrier function, partly by modulating the TJ proteins. However, the expression of TJ is dependent on both the refluxed material as well as the concentration of the bile salt.

The renin-angiotensin system in the esophageal mucosa of healthy subjects and patients with reflux disease.

OBJECTIVE: Components of the renin-angiotensin system (RAS) were recently discovered in the esophagus, which could be of interest in relation to gastroesophageal reflux disease (GERD). The present study was undertaken to confirm and further investigate the expression of RAS in healthy and refluxed exposed human esophageal mucosae. METHODS: Esophageal biopsies were obtained from healthy subjects (n = 34) and individuals with erosive reflux disease (ERD, n = 28). Evaluation of general morphology and histological signs of reflux as well as investigation of gene transcript, protein expression and localization of various RAS components using RT-PCR, ELISA, western blot and immunohistochemistry were performed. Physiological effects of the AT2R were investigated in Ussing chamber experiments. RESULTS: The study confirmed histological signs of reflux in ERD and expression of ACE, AT1R, AT2R and CatD in all examined specimens. In addition, the main effector peptide AngII, the pro-hormone AGT, the Mas receptor and the angiotensin-forming enzymes renin, CMA, CatG and NEP were present. Individuals with reflux disease had higher transcription activity of ACE and AT1R, increased protein levels of AT2R and lower levels of MasR. AT2R stimulation increased the ion currents in healthy epithelium, whereas epithelium from individuals with reflux disease exhibited no significant response. CONCLUSIONS: The study demonstrated that a local RAS is present in the human esophageal epithelium. Some RAS components were significantly altered in individuals diagnosed with ERD suggesting involvement in the pathophysiology of GERD.

ßHB inhibits glucose-induced GLP-1 secretion in GLUTag and human jejunal enteroids.

Ingestion of nutrients stimulates incretin secretion from enteroendocrine cells (EECs) of the epithelial layer of the gut. Glucagon-like peptide-1 (GLP-1) is one of these incretins that stimulate postprandial insulin release and signal satiety to the brain. Understanding the regulation of incretin secretion might open up new therapeutic options for obesity and type-2 diabetes mellitus. To investigate the inhibitory effect of the ketone body ß-hydroxybutyrate (ßHB) on glucose-induced GLP-1 secretion from EECs, in vitro cultures of murine GLUTag cells and differentiated human jejunal enteroid monolayers were stimulated with glucose to induce GLP-1 secretion. The effect of ßHB on GLP-1 secretion was studied using ELISA and ECLIA methods. GLUTag cells stimulated with glucose and ßHB were analysed using global proteomics focusing on cellular signalling pathways and the results were verified by Western blot. Results demonstrated ßHB had a significant inhibitory effect on glucose-induced GLP-1 secretion at a dose of 100 mM in GLUTag cells. In differentiated human jejunal enteroid monolayers, glucose-induced secretion of GLP-1 was inhibited at a much lower dose of 10 mM ßHB. The addition of ßHB to GLUTag cells resulted in decreased phosphorylation of kinase AKT and transcription factor STAT3 and also influenced the expressions of signalling molecule IRS-2, kinase DGKε and receptor FFAR3. In conclusion, ßHB displays an inhibitory effect on glucose-induced GLP-1 secretion in vitro in GLUTag cells and in differentiated human jejunal enteroid monolayers. This effect may be mediated through multiple downstream mediators of G-protein coupled receptor activation, such as PI3K signalling.

Antibodies to kidney endothelial cells contribute to a "leaky" glomerular barrier in patients with chronic kidney diseases.

Anti-endothelial cell antibodies (AECA) have been reported to cause endothelial dysfunction, but their clinical importance for tissue-specific endothelial cells is not clear. We hypothesized that AECA reactive with human kidney endothelial cells (HKEC) may cause renal endothelial dysfunction in patients with chronic kidney diseases. We report that a higher fraction (56%) of end-stage renal disease (ESRD) patients than healthy controls (5%) have AECA reactive against kidney endothelial cells (P <0.001). The presence of antibodies was associated with female gender (P < 0.001), systolic hypertension (P < 0.01), and elevated TNF-α (P < 0.05). These antibodies markedly decrease expression of both adherens and tight junction proteins VE-cadherin, claudin-1, and zonula occludens-1 and provoked a rapid increase in cytosolic free Ca(2+) and rearrangement of actin filaments in HKEC compared with controls. This was followed by an enhancement in protein flux and phosphorylation of VE-cadherin, events associated with augmented endothelial cell permeability. Additionally, kidney biopsies from ESRD patients with AECA but not controls demonstrated a marked decrease in adherens and tight junctions in glomerular endothelium, confirming our in vitro data. In summary, our data demonstrate a causal link between AECA and their capacity to induce alterations in glomerular vascular permeability.

In vitro assessment of epithelial electrical resistance in human esophageal and jejunal mucosae and in Caco-2 cell layers.

OBJECTIVE: There is a need for a technique allowing studies of human mucosal specimens collected during different clinical conditions. This study elucidates if square wave pulse analysis discriminates between epithelial and transmural electrical resistance and if there is an association with transepithelial permeability of molecular probes. METHODS: Mucosae from esophagus (surgical resections: n = 14; endoscopic biopsies: n = 15) and jejunum (n = 12) and Caco-2 cell monolayers were investigated in Ussing chambers. Transmural and epithelial electrical resistance were recorded by the use of standardized current pulses. Permeability was assessed using two fluorescein-labeled probes (weight 376 and 4000 Da). RESULTS: Baseline epithelial electrical resistance was higher in esophageal mucosa (~280 Ω*cm(2)), than in jejunal (~10 Ω*cm(2)) and Caco-2 cells (~140 Ω*cm(2)). The subepithelial contribution to the transmural resistance was higher in jejunal preparations (+88%) and Caco-2 cells (+75%), than in esophageal (+30%). During hypoxia the subepithelial resistance was unchanged, whereas the epithelial resistance decreased significantly in jejunal mucosa and Caco-2 cells. These findings coincided with increased transepithelial probe permeability and signs of disturbed morphology. Esophageal epithelia were resistant to hypoxia. However, exposure to deoxycholic acid and trypsin abolished the esophageal epithelial resistance and increased probe permeability. Endoscopic esophageal biopsies from patients with erosive reflux disease exhibited significantly lower epithelial resistance and higher current than healthy subjects. CONCLUSION: Square wave pulse analysis in Ussing chambers is suitable for assessment of epithelial electrical resistance that can reflect transepithelial permeability of molecular probes with known size. Moreover, the technique discriminated between healthy and reflux-diseased esophageal mucosal biopsies.