A novel model of adenine-induced chronic kidney disease-associated gastrointestinal dysfunction in mice: The gut-kidney axis.

Although constipation is a common complication of chronic kidney disease (CKD), there is no animal model that can be used to study the association between renal impairment and gastrointestinal function without interfering with the gastrointestinal tract of the model. Therefore, we determined whether adenine could induce CKD in association with gastrointestinal dysfunction. Six-week-old ICR mice were intraperitoneally injected with saline, 25, 50, or 75 mg adenine/kg body weight for 21 days. Blood urea nitrogen (BUN), plasma creatinine, and renal histopathology were evaluated. Defecation status was evaluated from defecation frequency and fecal water content. Colonic smooth muscle contraction was measured by the organ bath technique, and transepithelial electrical resistance (TEER) was measured using an Ussing chamber. In the 50 mg/kg treatment group, BUN and creatinine were significantly increased compared with control, and inflammatory cell infiltration, glomerular necrosis, tubular dilatation, and interstitial fibrosis were observed in renal tissues. Mice in this group also showed a significant decrease in defecation frequency, fecal water content, colonic motility index, and TEER. Overall, 50 mg/kg of adenine was the best dose to induce CKD with associated constipation and intestinal barrier impairment. Therefore, this adenine administration model can be recommended for CKD-associated gastrointestinal dysfunction research.

Probiotics of Lacticaseibacillus paracasei SD1 and Lacticaseibacillus rhamnosus SD11 attenuate inflammation and ß-cell death in streptozotocin-induced type 1 diabetic mice.

Probiotics provide health benefits in various aspects and are believed to modulate the immune system by balancing gut microbiota homeostasis, termed the "microbiota-immune axis". Recent evidence supports that several Lactobacillus strains possess glucose-lowering and anti-inflammatory effects in an animal model of type 1 diabetes (T1D). Although probiotics of Lacticaseibacillus paracasei SD1 (SD1) and Lacticaseibacillus rhamnosus SD11 (SD11) exert human oral health benefits by reducing harmful bacterial populations, their clinical application regarding hypoglycemic-related traits as well as the underlying mechanisms are still lacking. In this report, we used multiple low doses of streptozotocin (STZ)-induced diabetic BALB/c mice to explore the effects of SD1 and SD11 supplementation on the regulation of markers related to T1D. Experimental mice were randomly assigned into five groups, non-STZ + V, STZ + V, STZ + SD1, STZ + SD11, and STZ + SDM (mixture of SD1 and SD11), and physiological data were measured every week. Blood and pancreas samples were collected at 4- and 8-weeks. Our results indicate that supplementation with SD1, SD11, or SDM for 8 weeks significantly improved body weights, glycemic levels, glucose tolerance, insulin levels, and lipid profiles. Probiotic administration also preserved islet integrity and increased ß-cell mass in STZ-injected mice, as well as prevented infiltration of macrophages, CD4+, and CD8+ T cells into the islets. Significantly, SD1 and SD11 suppressed the levels of IL1-ß, TNF-α and IFN-γ and increased IL-10, which is concomitant with the inhibition of cleaved caspase 3, caspase 9, caspase 8, proapoptotic Bax, NF-κBp65, pSTAT1, and iNOS. Additionally, the survival ability of ß-cells was mediated by upregulated anti-apoptotic Bcl2. We conclude that SD1 and SD11 attenuate STZ-induced diabetic mice by stabilizing glycemic levels and reducing inflammation, thereby protecting ß-cells. Among the probiotic treatment groups, SD11 revealed the best results in almost all parameters, indicating its potential use for alleviating hyperglycemia-associated symptoms.

Sargassum plagiophyllum Extract Enhances Colonic Functions and Modulates Gut Microbiota in Constipated Mice.

Constipation is a symptom that is widely found in the world's population. Various dietary supplementations are used to relieve and prevent constipation. Seaweed is widely used for its health benefits. In this study, we aimed to investigate the effects of Sargassum plagiophyllum extract (SPE) on functions of the gastrointestinal tract and gut microbiota. The results show that SPE pretreatment increased the frequency of gut contraction, leading to reduce gut transit time. SPE pretreatment also significantly increased the secretion of Cl− and reduced Na+ absorption, increasing fecal water content in constipated mice (p < 0.05). In addition, the Bifidobacteria population in cecal contents was significantly higher in constipated mice pretreated with 500 mg/kg SPE for 14 days than in untreated constipated mice (p < 0.05). Our findings suggest that SPE can prevent constipation in loperamide-induced mice. This study may be useful for the development of human food supplements from S. plagiophyllum, which prevent constipation.

Effects of standardized [6]-gingerol extracts and [6]-gingerol on isolated ileum and lower esophageal sphincter contractions in mice.

Standardized [6]-gingerol extracts were prepared by microwave-assisted extraction using 20% v/v glycerin in ethanol and 20% v/v eutectic mixture of sucrose and citric acid in ethanol as alternative green solvents. The extracts obtained from 20% v/v glycerin in ethanol (GEE) and 20% v/v eutectic mixture of sucrose and citric acid in ethanol (EMSCEE) were standardized by HPLC to contain 17.0 mg/g of [6]-gingerol. The effects of the extracts on mouse ileal contractions via M3 and 5-HT3 receptors as well as lower esophageal sphincter (LES) contraction were determined in vitro relative to the marker compound, [6]-gingerol. [6]-Gingerol, GEE and EMSCEE demonstrated significant and concentration-dependent inhibitory effects on ileal contraction in mice via M3 and 5-HT3 receptors in a noncompetitive manner. In addition, [6]-gingerol and EMSCEE tend to increase the LES tone. These results indicated the potential of GEE and EMSCEE to attenuate nausea and vomiting and might be used as nutraceuticals.

Amelioration of gut dysbiosis and gastrointestinal motility by konjac oligo-glucomannan on loperamide-induced constipation in mice.

OBJECTIVE: Konjac oligo-glucomannan (KOG) is a non-digestible dietary fiber that is resistant to digestion and absorption in gastrointestinal (GI) tract. Thus, it might be used as an alternative management for constipation. The aim of this study was to evaluate the effects of KOG on gut motility and microbiota to relieve constipation in mice. METHODS: Mice received Bifidobacterium animalis, lactulose, konjac glucomannan (KGM), or KOG for 14 d. Constipation was induced by 5 mg/kg loperamide days 12 through 14 in all groups except the control. Defecation frequency, small intestinal transit, and total gut transit time were indicated by counting the number of feces, and using charcoal meal and Evans blue as markers, respectively. Smooth muscle (SM) contraction and gut motility were evaluated by organ bath and GI motility monitor system. Gut microbiota were measured by fluorescence in situ hybridization technique. RESULTS: KOG significantly (P < 0.01) increased defecation frequency and small intestinal transit but decreased total gut transit time when compared with the constipation-without-treatment group. These results were similar to the effects of Bifidobacterium animalis, lactulose, and KGM. KOG ameliorated the effect of loperamide on contraction frequency of distal colonic circular SM. The motility patterns were changed in the KOG group from non-propagation to propagation contraction. KOG significantly inhibited the effects of loperamide on gut microbiota by increasing the numbers of Bifidobacterium spp. and decreasing the numbers of Clostridium spp. and Bacteroides spp. CONCLUSION: These results suggest that KOG acts as a prebiotic and stimulant laxative for relief and prevention of constipation.

Diarylheptanoid 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one enhances C2C12 myoblast differentiation by targeting membrane estrogen receptors and activates Akt-mTOR and p38 MAPK-NF-κB signaling axes.

Diarylheptanoid, 1-(4-hydroxyphenyl)-7-phenyl(6E)-6-hepten-3-one (HPPH), has been reported to enhance myoblast differentiation via estrogen receptor (ER). However, the underlying signaling pathway promising this action remains unknown. The present study thus aimed to investigate the signaling pathway of HPPH that enhances myoblast differentiation. Confluence C2C12 myoblasts were induced to differentiate in the absence or presence of HPPH (10 nM). Differentiation markers (myosin heavy chain (MHC) and myogenin) and other signaling molecules implicated in myogenic differentiation were analyzed by immunostaining and western blotting methods. To identify the location of ER and the signaling molecules, specific inhibitors were applied targeting these molecules. Nuclear factor-κB (NF-κB) DNA binding activity was measured using the electrophoresis mobility shift assay. The results showed that HPPH enhanced myoblast differentiation by increasing MHC and myogenin levels, number, and size as well as the fusion index of myotubes. These actions occurred via membrane ER. Several MAPK proteins were activated at the early stage of differentiation. However, only Akt and p38 MAPK, but not ERK, were implicated in these effects. The underlying signaling molecules of Akt to enhance myogenic differentiation by HPPH, at least in part, were mTOR/P70S6K and GSK-3ß. On the other hand, the downstream signaling molecule of p38 MAPK was NF-κB. Our results suggested that HPPH enhanced myogenic differentiation by binding with membrane ER, which in turn recruited multiple axes including Akt-mTOR-P70S6K, Akt-GSK-3ß, and p38 MAPK-NF-κB.

Prebiotic oligosaccharides from dragon fruits alter gut motility in mice.

Dragon fruit oligosaccharide (DFO) has a prebiotic property which improves gut health by selectively stimulating the colonic microbiota. Altering microbiota composition may affect intestinal motility. However, no study has been done to understand the DFO effects on gut motor functions. This research thus aimed to investigate the DFO effects on mice colons compared to the prebiotic fructo-oligosaccharide (FOS) and probiotic bifidobacteria. The mice in this study received distilled water; 100, 500, and 1000 mg/kg DFO; 1000 mg/kg FOS; or 109 CFU Bifidobacterium animalis daily for 1 week and some treatments for 2 weeks. Gastrointestinal transits were analysed and motility patterns, smooth muscle (SM) contractions and morphological structures of the colons were assessed. Administration of FOS, 500 and 1000 mg/kg DFO significantly increased fecal output when compared to the control group. In mice treated with FOS and bifidobacteria, gut transit time was reduced, while upper gut transit was increased in comparison to DFO groups. Spatiotemporal maps of colonic wall motions showed that DFO increased the number of colonic non-propagation contractions and fecal pellet velocity, consistent with the results from groups treated with FOS and bifidobacteria. DFO also increased the amplitude and duration of colonic SM contractions. Histological stains showed normal epithelia, crypts, goblet cells, and SM thickness in all groups. In conclusion, DFO increased colonic SM contractions without morphological change and acted as a bulk-forming and stimulant laxative to increase fecal output and intestinal motility. Thus, DFO as a dietary supplement may promote gut health and correct gastrointestinal motility disorders.

Furosemide suppresses ileal and colonic contractility via interactions with GABA-A receptor in mice.

The loop diuretic furosemide has an action to inhibit Na+ -K+ -2Cl- co-transporter at the thick ascending limb of Henle's loop resulting in diuresis. Furosemide also has the non-diuretic effects by binding to GABA-A receptor which may involve the gastrointestinal tract. The aim of this study was to investigate the effects of furosemide on smooth muscle contractions in mice ileum and proximal colon. Each intestinal segment suspended in an organ bath was connected to a force transducer. Signal output of mechanical activity was amplified and recorded for analysis using PowerLab System. After equilibration, the intestine was directly exposed to furosemide, GABA, GABA-A receptor agonist (muscimol), or muscarinic receptor antagonist (atropine). Furosemide (50, 100 and 500 µmol L-1 ) acutely reduced the amplitude of ileal and colonic contraction. In the ileum, 1 mmol L-1 GABA and 10-60 µmol L-1 muscimol significantly increased the amplitude, whereas in the colon, 50-100 mmol L-1 GABA and 60 µmol L-1 muscimol decreased the contractions. The contractions were also significantly suppressed by atropine. To investigate the mechanisms underlying the inhibiting effect of furosemide, furosemide was added to the organ bath prior to the addition of muscimol or atropine. A comparison of furosemide combined with muscimol or atropine group and furosemide group showed no significant difference of the ileal contraction, but the amplitude of colonic contraction significantly decreased when compared to adding furosemide alone. These results suggest that furosemide can reduce the ileal and proximal colonic contraction mediated by blocking and supporting of GABA-A receptor, respectively, resulting in decreased acetylcholine release.

Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating.

KEY POINTS: Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. ABSTRACT: Cross-species comparative studies are a powerful approach to understanding the epithelial Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl(-) channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl(-) currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl(-) channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.

Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics.

BACKGROUND AND PURPOSE: Loop diuretics are widely used to inhibit the Na(+), K(+), 2Cl(-) co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade. EXPERIMENTAL APPROACH: Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR. KEY RESULTS: When added to the intracellular solution, loop diuretics inhibited CFTR Cl(-) currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl(-) channels with 'very fast' speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with 'intermediate' speed and drug-induced blocking events were distinct from channel closures. CONCLUSIONS AND IMPLICATIONS: Structure-activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity.

Fibroblast growth factor-23 negates 1,25(OH)2D3-induced intestinal calcium transport by reducing the transcellular and paracellular calcium fluxes.

The calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has been known to stimulate intestinal calcium transport via both transcellular and paracellular pathways. Recently, we reported that the 1,25(OH)2D3-enhanced calcium transport in the mouse duodenum could be abolished by fibroblast growth factor (FGF)-23, but the targeted calcium transport pathway has been elusive. Herein, the 1,25(OH)2D3-enhanced calcium transport was markedly inhibited by FGF-23 and inhibitors of the basolateral calcium transporters, NCX1 and PMCA1b, suggesting the negative effect of FGF-23 on the transcellular calcium transport. Similar results could be observed in the intestinal epithelium-like Caco-2 monolayer. Although the Arrhenius plot indicated that FGF-23 decreased the potential barrier (e.g., activation energy) of the paracellular calcium movement, FGF-23 was found to modestly decrease the 1,25(OH)2D3-enhanced paracellular calcium transport and calcium permeability. Moreover, FGF-23 affected the 1,25(OH)2D3-induced change in duodenal water permeability as determined by tritiated water, but both 1,25(OH)2D3 and FGF-23 were without effects on the transepithelial fluxes of paracellular markers, (3)H-mannitol and (14)C-polyethylene glycol. It could be concluded that FGF-23 diminished the 1,25(OH)2D3-enhanced calcium absorption through the transcellular and paracellular pathways. Our findings have thus corroborated the presence of a bone-kidney-intestinal axis of FGF-23/vitamin D system in the regulation of calcium homeostasis.

1,25-Dihydroxyvitamin D3 -induced intestinal calcium transport is impaired in ß-globin knockout thalassemic mice.

Besides being a common haematological disorder caused by a reduction in ß-globin production, ß-thalassemia has been reported to impair body calcium homeostasis, leading to massive bone loss and increased fracture risk. Here, we demonstrated that heterozygous ß-globin knockout thalassemic mice had a lower rate of duodenal calcium absorption compared with the wild-type littermates, whereas the epithelial electrical parameters, including transepithelial resistance, were not affected, suggesting no change in the epithelial integrity and permeability. Daily subcutaneous injection of 1 µg kg(-1) 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] for 3 days enhanced the duodenal calcium absorption in wild-type, but not in thalassemic mice. Although ß-thalassemia increased the mRNA level of divalent metal transporter-1, an iron transporter in the duodenum, it had no effect on the transcripts of ferroportin-1 or the principal calcium transporters. In conclusion, ß-thalassemia impaired the 1,25(OH)2 D3 -dependent intestinal calcium absorption at the post-transcriptional level, which, in turn, contributed to the dysregulation of body calcium metabolism and ß-thalassemia-induced osteopenia.

Fibroblast growth factor-23 abolishes 1,25-dihydroxyvitamin D3-enhanced duodenal calcium transport in male mice.

Despite being widely recognized as the important bone-derived phosphaturic hormone, whether fibroblast growth factor (FGF)-23 modulated intestinal calcium absorption remained elusive. Since FGF-23 could reduce the circulating level of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], FGF-23 probably compromised the 1,25(OH)2D3-induced intestinal calcium absorption. FGF-23 may also exert an inhibitory action directly through FGF receptors (FGFR) in the intestinal cells. Herein, we demonstrated by Ussing chamber technique that male mice administered 1 µg/kg 1,25(OH)2D3 sc daily for 3 days exhibited increased duodenal calcium absorption, which was abolished by concurrent intravenous injection of recombinant mouse FGF-23. This FGF-23 administration had no effect on the background epithelial electrical properties, i.e., short-circuit current, transepithelial potential difference, and resistance. Immunohistochemical evidence of protein expressions of FGFR isoforms 1-4 in mouse duodenal epithelial cells suggested a possible direct effect of FGF-23 on the intestine. This was supported by the findings that FGF-23 directly added to the serosal compartment of the Ussing chamber and completely abolished the 1,25(OH)2D3-induced calcium absorption in the duodenal tissues taken from the 1,25(OH)2D3-treated mice. However, direct FGF-23 exposure did not decrease the duodenal calcium absorption without 1,25(OH)2D3 preinjection. The observed FGF-23 action was mediated by MAPK/ERK, p38 MAPK, and PKC. Quantitative real-time PCR further showed that FGF-23 diminished the 1,25(OH)2D3-induced upregulation of TRPV5, TRPV6, and calbindin-D(9k), but not PMCA(1b) expression in the duodenal epithelial cells. In conclusion, besides being a phosphatonin, FGF-23 was shown to be a novel calcium-regulating hormone that acted directly on the mouse intestine, thereby compromising the 1,25(OH)2D3-induced calcium absorption.