Chloride Secretion Research Articles

Synthesis of ionizable lipopolymers using split-Ugi reaction for pulmonary delivery of various size RNAs and gene editing.

We present an efficient approach for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to rapidly create a library of complex functional ionizable lipopolymers. We synthesized a diverse library of 155 polymers, formulated them into polyplexes to establish structure-activity relationships crucial for endosomal escape and efficient transfection. After discovering a lead structure, lipopolymer-lipid hybrid nanoparticles are introduced to preferentially deliver to and elicit effective mRNA transfection in lung endothelium and immune cells, including T cells with low in vivo toxicity. The lipopolymer-lipid hybrid nanoparticles showed 300-fold improvement in systemic mRNA delivery to the lung compared to in vivo -JetPEI ® . Lipopolymer-lipid hybrid nanoparticles demonstrated efficient delivery of mRNA-based therapeutics for treatment of two different disease models. Lewis Lung cancer progression was significantly delayed after treatment with loaded IL-12 mRNA in U155@lipids after repeated i.v. administration. Systemic delivery of human CFTR (hCFTR) mRNA resulted in production of functional form of CFTR protein in the lungs. The functionality of hCFTR protein was confirmed by restoration of CFTR- mediated chloride secretion in conductive airway epithelia in CFTR knockout mice after nasal instillation of hCFTR mRNA loaded U155@lipids. We further showed that, U155@lipids nanoparticles can deliver complex CRISPR-Cas9 based RNA cargo to the lung, achieving 5.6 ± 2.4 % gene editing in lung tissue. Moreover, we demonstrated successful PD-1 gene knockout of T cells in vivo . Our results highlight a versatile delivery platform for systemic delivering of mRNA of various sizes for gene therapy for a variety of therapeutics.

Recent Advances in the Treatment of Irritable Bowel Syndrome: Addressing Diarrhea, Constipation, and Abdominal Pain

Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder characterized by abdominal pain, bloating, and changes in bowel habits. This paper aim was to provide an overview of recent advancements in the treatment of IBS, focusing on the management of its main symptoms: diarrhea, constipation, and abdominal pain. For individuals with IBS-D, various therapeutic options are available, including rifaximin, peripheral opioid agonists, mixed opioid agonists/antagonists, bile acid sequestrants, and serotonin 5-HT3 receptor antagonists. Rifaximin, a nonabsorbable antibiotic, has demonstrated significant effectiveness in reducing IBS symptoms, such as bloating and loose stools, with good tolerability and safety. Also the repeated use of rifaximin for managing recurrent IBS symptoms. In IBS-C patients, bulking agents, osmotic laxatives, and prokinetic agents like lubiprostone have shown efficacy in improving constipation and overall symptoms. Linaclotide, a drug that increases intestinal chloride secretion, has demonstrated significant improvement in bowel movements and abdominal pain in IBS-C patients. However, it may cause diarrhea in some individuals. IBS-M, characterized by alternating constipation and diarrhea, poses a challenge in terms of specific drug treatments. A comprehensive assessment of potential underlying causes and careful history taking is crucial for effective management. Abdominal pain is a common symptom in IBS, regardless of subtype. Antispasmodic drugs, peppermint oil, and trimebutine have shown effectiveness in reducing pain and improving symptoms. Antidepressants, such as tricyclic antidepressants and selective serotonin reuptake inhibitors, may offer relief by modulating pain pathways and influencing gastrointestinal motility. Benzodiazepines, particularly dextofisopam, have shown potential in improving stool consistency but require further evaluation. In conclusion, recent advancements in the treatment of IBS have provided a range of therapeutic options targeting its main symptoms. However, further research is needed to develop tailored treatments for specific IBS subtypes and to explore the efficacy of potential alternatives, including benzodiazepines. Improved management of IBS symptoms can significantly enhance the quality of life for affected individuals.

P2Y2R and Cyst Growth in Polycystic Kidney Disease.

Key Points Polycystic kidney disease (PKD) is characterized by continuous cyst growth, which results in a decline in kidney function.Deletion of P2Y2R and pharmacological antagonism of purinergic signaling significantly reduced cyst growth in an orthologous PKD mouse model.P2Y2R was expressed in cysts of human PKD nephrectomies, which makes P2Y2R a reasonable target for treatment of PKD. Background Autosomal dominant polycystic kidney disease (ADPKD) is characterized by multiple bilateral kidney cysts that gradually enlarge, resulting in a decline in kidney function. Cyst growth is significantly driven by ATP-dependent chloride secretion mediated by the ion channel TMEM16A. This pathway is further augmented in advanced stages of the disease by hypoxia and activation of hypoxia-inducible factor (HIF)-1α. The mechanisms by which ATP leads to activation of TMEM16A and how HIF-1α contributes to cyst growth in vivo have remained elusive. Methods Mice with an inducible tubule-specific deletion of Pkd1 were compared with mice with an additional codeletion of the purinergic receptor P2y2r. Furthermore, animals were challenged by pharmacological activation of HIF-1α and Pkd1-deficient mice were treated with suramin, an antagonist of purinergic signaling. In addition, expression of P2Y2R, TMEM16A, and HIF-1α was analyzed in nephrectomy samples from 27 patients with ADPKD. Results Genetic deletion of P2y2r significantly inhibited cyst growth in vivo. In addition, aggravation of the polycystic phenotype mediated by pharmacological activation of HIF-1α was reduced by deletion of P2y2r. Application of suramin to pharmacologically inhibit purinergic signaling also suppressed cyst enlargement in vivo. Analysis of kidney samples from 27 patients with ADPKD revealed significant expression of P2Y2R at the luminal site of the cyst-lining epithelium. Conclusions P2Y2R was significantly expressed in human and mouse polycystic kidneys. Deletion and antagonism of P2Y2R reduced cyst enlargement in an ADPKD mouse model.

Tuft cell-derived acetylcholine promotes epithelial chloride secretion and intestinal helminth clearance

Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.

Purinergic Signaling Drives Multiple Aspects of Rotavirus Pathophysiology

Background: Rotavirus causes life-threatening diarrhea in children, resulting in ~200,000 deaths each year. Rotavirus infects a limited number of cells at the tips of the villi in the small intestine; however, rotavirus is known to dysregulate cells distant from the site of infection through paracrine signaling. We recently identified that rotavirus infected cells release the purinergic signaling molecule ADP, which binds to the P2Y1 receptor on nearby uninfected cells; causing a signaling cascade that affects distant uninfected cells. In vitro P2Y1 was found to contribute to chloride and serotonin secretion; suggesting P2Y1 may activate pathways responsible for diarrhea. Hypothesis: We hypothesize that P2Y1 orchestrates cell-cell communication involved in diarrhea and infection severity. Methods: To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection, we used the mouse-like rotavirus (rD6/2) to investigate the effects of purinergic signaling in the context of homologous murine rotavirus infection in vivo. C57Black6 mouse pups were orally gavaged rD6/2 rotavirus at day 4-6 of age and assessed over the course of 5 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4mg/kg MRS2500, a P2Y1-selective, competitive antagonist. Stool was collected and scored for diarrhea daily prior to each treatment. Pups were euthanized and small intestine tissue was collected at 3- and 5-days post infection for immunostaining, qRT-PCR and luminal contents. Intestinal organoids derived from Balb/C pup jejunum were inoculated with rD6/2 rotavirus, followed by supplementation with vehicle or MRS2500 to assess fluid secretion by organoid swelling over time. Results: Rotavirus-infected pups exhibited significant diarrhea incidence and high diarrhea scores on days 2, 3, and 4 post infection. Treatment of rotavirus-infected mouse pups with MRS2500 resulted in decreased severity and incidence of diarrhea compared to vehicle treated controls. This was consistent with in vitro, rD6/2-infected organoids exhibiting a significant increase in organoid swelling compared to uninfected organoids, which was significantly prevented by inhibition of P2Y1 signaling. Viral stool shedding from MRS2500 treated pups was significantly lower than saline-treated pups starting at day 4 post infection, which suggests P2Y1 signaling may amplify rotavirus replication in vivo. Finally, transmission experiments in litters with infected (but not treated) and pretreated MRS2500 uninfected pups show a decreased incidence in diarrhea suggesting that P2Y1 inhibition has a potential protective role in reducing the effects of transmission. Conclusion: Collectively these findings establish a conserved role of purinergic signaling in the pathophysiology of rotavirus infection. Additionally, our data indicates P2Y1 as a new candidate for host-targeted therapeutics that could have both antiviral and antidiarrheal effects against rotavirus pathophysiology. R01 DK115507 (Hyser) R01 AI158683 (Hyser)APS Postdoctoral Fellowship (Engevik) F32 DK130288 (Engevik). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Apical pH regulates calcium-activated chloride secretion across human airway epithelia

Anion secretion across airway epithelia contributes to host defense by increasing airway surface liquid (ASL) volume and pH. Anion channels in the apical membrane of airway epithelia include Ca2+-activated Cl− channels (CaCCs) and cystic fibrosis transmembrane conductance regulator (CFTR) channels. Mutations in the CFTR gene that encode nonfunctional CFTR channels cause cystic fibrosis (CF); thus, activating CaCCs may restore anion secretion in people with CF. ATP secreted into the ASL activates purinergic receptors, leading to an increase in intracellular Ca2+ (Cai2+) and the opening of CaCCs. Data from docking simulations suggest that the protonation state of ATP influences its affnity for purinergic receptors, therefore we tested the hypothesis that ASL pH influences the effcacy of ATP to activate CaCCs in airway epithelia. In saline solutions buffered with 5 mM ATP, titration experiments revealed a pKa of ~6.9 at 37°C. Thus, ATP exists in its protonated ATP3− and deprotonated ATP4− in ASL that can range from pH 6 to pH 7.5. To determine the effect of pH on ATP-stimulated Cl− secretion through CaCCs, we measured Cl− secretion across CF epithelia generated by several ATP concentrations with apical pH-6.00, -6.70, and -7.40 solutions. As pH became alkaline, the EC50 for the relationship between [ATP] and Cl− secretion decreased. Using the Henderson-Hasselbalch equation and the pKa for ATP, we determined the relationship between ATP4− and Cl− secretion. This analysis revealed that regardless of apical pH values, the relationship between [ATP4−] and Cl− secretion aligned to a single experimental fit. These data suggest that pH regulates the CaCC response by deprotonating ATP. To examine the pH dependence of purinergic receptor activation, we measured ATP-induced Cai2+ responses in acutely dissociated human airway epithelial cells loaded with FURA-2 and perfused with pH-7.40 or -6.50 solutions. Separate experiments measuring intracellular pH in pH-7.40 or 6.50 solutions informed Kd values for FURA-2 experiments. With an [ATP] that induces similar levels of Cl− secretion from pH 6.00 to pH 7.40 (100 μM ATP), we observed similar Cai2+ responses for pH-7.40 and -6.50 solutions. However, when we repeated the experiment with an [ATP] that induces pH-sensitive Cl− secretion (10 μM ATP), we observed larger Ca2+ responses with pH-7.40 solutions than with pH-6.50 solutions. These data indicate that pH influences purinergic receptor activation near the pKa of ATP, a finding consistent with ATP4− activating purinergic receptors. These data have implications in CF and airway inflammation. In CF, decreased CFTR-mediated HCO−3 secretion acidifies the ASL, thus a large portion of ATP will exist in the protonated ATP3− species requiring a higher total [ATP] to open CaCCs. Inflammatory cytokines that cause airway inflammation increase the ASL pH, therefore a larger portion of ATP will exist in the deprotonated ATP4− species requiring less total [ATP] to open CaCCs. Cystic Fibrosis Foundation Research Development Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluating the Efficacy of Prucalopride, a 5-HT4 Agonist, in Managing Antipsychotic-Induced Constipation: A Prospective Randomized Controlled Trial Conducted at Chronic Psychiatric Rehabilitation Facilities on Corfu Island

Introduction Achieving successful stabilization in patients with mental disorders often requires the administration of multiple antipsychotic medications, with the increasing prevalence of clozapine in cases resistant to other treatments. Constipation emerges as a particularly troublesome side effect, gradually progressing into a chronic state of gastrointestinal dysfunction, often accompanied by recurrent episodes of paralytic ileus of varying severity. Prucalopride, a 5-HT4 agonist, selectively targets receptors within the intestinal system.This interaction induces muscular contractions and promotes chloride secretion. Literature suggest its potential efficacy in managing constipation induced by clozapine. In light of these observations, we designed and will conduct a randomized controlled trial to evaluate the effectiveness of prucalopride in alleviating constipation in patients who had shown limited responsiveness to conventional laxatives or other conservative treatmentsObjectivesThe primary objective of this article is to present the methodology of a randomized control trial assessing the efficacy of prucalopride in the treatment of constipation among patients with mental disordersMethodsThe study will enroll 60 adult patients with mental disorders who will require more than two antipsychotic medications, including clozapine, for stabilization, and who will be experiencing constipation as a side effectTo ensure the validity of the study, the following additional inclusion criteria will be applied: -Patients will have no severe acute medical conditions-Patients will have no history of malignancy-Patients will have no severe respiratory or cardiac diseases-Patients will have negative results from an endoscopic evaluation of the large bowel, ruling out conditions such as irritable bowel syndrome, ischemic colitis, inflammatory bowel disease, or malignant neoplastic diseaseFollowing the screening process, the patients will be randomly assigned to one of two treatment groups:Prucalopride Group: Patients in this group will receive prucalopride for the treatment of refractory constipationConservative Treatment Group: Patients in this group will continue with conservative treatments.The treatment’s success will be determined based on specific endpoints:-Normalization of bowel movements, characterized by having more than five bowel movements per week- Resolution of symptoms related to gastrointestinal dysfunction, including pain, bloating, defecation difficulties, and paralytic ileus ResultsFollowing the conclusion of the study, data from both groups will be meticulously collected and subjected to rigorous statistical analysis to identify differences in treatment outcomes between these two therapeutic approachsConclusionsThe detailed findings will be presented in a forthcoming articleDisclosure of InterestNone Declared

Adenosine in Intestinal Epithelial Barrier Function.

At the intestinal front, several lines of defense are in place to resist infection and injury, the mucus layer, gut microbiome and strong epithelial junctions, to name a few. Their collaboration creates a resilient barrier. In intestinal disorders, such as inflammatory bowel disease (IBD), barrier function is compromised, which results in rampant inflammation and tissue injury. In response to the destruction, the intestinal epithelium releases adenosine, a small but powerful nucleoside that functions as an alarm signal. Amidst the chaos of inflammation, adenosine aims to restore order. Within the scope of its effects is the ability to regulate intestinal epithelial barrier integrity. This review aims to define the contributions of adenosine to mucus production, microbiome-dependent barrier protection, tight junction dynamics, chloride secretion and acid-base balance to reinforce its importance in the intestinal epithelial barrier.

Structural identification of a selectivity filter in CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that regulates transepithelial salt and fluid homeostasis. CFTR dysfunction leads to reduced chloride secretion into the mucosal lining of epithelial tissues, thereby causing the inherited disease cystic fibrosis. Although several structures of CFTR are available, our understanding of the ion-conduction pathway is incomplete. In particular, the route that connects the cytosolic vestibule with the extracellular space has not been clearly defined, and the structure of the open pore remains elusive. Furthermore, although many residues have been implicated in altering the selectivity of CFTR, the structure of the "selectivity filter" has yet to be determined. In this study, we identify a chloride-binding site at the extracellular ends of transmembrane helices 1, 6, and 8, where a dehydrated chloride is coordinated by residues G103, R334, F337, T338, and Y914. Alterations to this site, consistent with its function as a selectivity filter, affect ion selectivity, conductance, and open channel block. This selectivity filter is accessible from the cytosol through a large inner vestibule and opens to the extracellular solvent through a narrow portal. The identification of a chloride-binding site at the intra- and extracellular bridging point leads us to propose a complete conductance path that permits dehydrated chloride ions to traverse the lipid bilayer.

Pro-inflammatory cytokines stimulate CFTR-dependent anion secretion in pancreatic ductal epithelium.

Loss of CFTR-dependent anion and fluid secretion in the ducts of the exocrine pancreas is thought to contribute to the development of pancreatitis, but little is known about the impact of inflammation on ductal CFTR function. Here we used adult stem cell-derived cell cultures (organoids) obtained from porcine pancreas to evaluate the effects of pro-inflammatory cytokines on CFTR function. Organoids were cultured from porcine pancreas and used to prepare ductal epithelial monolayers. Monolayers were characterized by immunocytochemistry. Epithelial bicarbonate and chloride secretion, and the effect of IL-1β, IL-6, IFN-γ, and TNF-α on CFTR function was assessed by electrophysiology. Immunolocalization of ductal markers, including CFTR, keratin 7, and zonula occludens 1, demonstrated that organoid-derived cells formed a highly polarized epithelium. Stimulation by secretin or VIP triggered CFTR-dependent anion secretion across epithelial monolayers, whereas purinergic receptor stimulation by UTP, elicited CFTR-independent anion secretion. Most of the anion secretory response was attributable to bicarbonate transport. The combination of IL-1β, IL-6, IFN-γ, and TNF-α markedly enhanced CFTR expression and anion secretion across ductal epithelial monolayers, whereas these cytokines had little effect when tested separately. Although TNF-α triggered apoptotic signaling, epithelial barrier function was not significantly affected by cytokine exposure. Pro-inflammatory cytokines enhance CFTR-dependent anion secretion across pancreatic ductal epithelium. We propose that up-regulation of CFTR in the early stages of the inflammatory response, may serve to promote the removal of pathogenic stimuli from the ductal tree, and limit tissue injury.

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.

Postbiotic Preparation of Lacticaseibacillus rhamnosus GG against Diarrhea and Oxidative Stress Induced by Spike Protein of SARS-CoV-2 in Human Enterocytes.

The Spike protein of SARS-CoV-2 acts as an enterotoxin able to induce chloride secretion and production of reactive oxygen species (ROS), involved in diarrhea pathogenesis. L. rhamnosus GG (LGG) is recommended in pediatric acute gastroenteritis guidelines as a therapy independent of infectious etiology. We tested a postbiotic preparation of LGG (mLGG) in an in vitro model of COVID-associated diarrhea. Caco-2 cell monolayers mounted in Ussing chambers were exposed to Spike protein, and electrical parameters of secretory effect (Isc and TEER) were recorded in the Ussing chambers system. Oxidative stress was analyzed by measuring ROS production (DCFH-DA), GSH levels (DNTB), and lipid peroxidation (TBARS). Experiments were repeated after mLGG pretreatment of cells. The Isc increase induced by Spike was consistent with the secretory diarrhea pattern, which was dependent on oxidative stress defined by a 2-fold increase in ROS production and lipid peroxidation and variation in glutathione levels. mLGG pretreatment significantly reduced the secretory effect (p = 0.002) and oxidative stress, namely ROS (p < 0.001), lipid peroxidation (p < 0.001), and glutathione level changes (p < 0.001). LGG counteracts Spike-induced diarrhea by inhibiting the enterotoxic effect and oxidative stress. The LGG efficacy in the form of a postbiotic depends on metabolites secreted in the medium with antioxidant properties similar to NAC. Because SARS-CoV-2 is an enteric pathogen, the efficacy of LGG independent of etiology in the treatment of acute gastroenteritis is confirmed by our data.

Puzzle resolved: CFTR mediates chloride homeostasis by segregating absorption and secretion to different cell types.

In the lungs, the cystic fibrosis transmembrane conductance regulator (CFTR) regulates ion transport in surface-airway epithelia and submucosal glands, thus determining airway surface liquid (ASL) volume and mucus hydration. In this issue of the JCI, Lei Lei and colleagues report that the CFTR-rich and barttin/Cl- channel-expressing ionocytes mediate chloride absorption across airway epithelia, whereas the more abundant basal cells and secretory cells mediate chloride secretion. Thus, CFTR-mediated secretion and absorption of chloride ions in the lung are segregated by cell type, which has implications for future molecular therapies for cystic fibrosis lung disease.

Patient-derived enteroids provide a platform for the development of therapeutic approaches in microvillus inclusion disease.

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.

222 Signaling mechanisms driving CFTR-independent chloride secretion in Calu-3 cells

222 Signaling mechanisms driving CFTR-independent chloride secretion in Calu-3 cells

In-vivo Evaluation of Nephroprotective Activity of Naringenin against ADPKD and MDCK-derived Cysts

Aim: Determining the in-vivo assessment of Naringenin’s nephroprotective efficacy against Autosomal dominant polycystic kidney disease (ADPKD) and Madin-Darby canine kidney (MDCK) derived cysts. Method: Our research on the molecular mechanism of naringenin, a flavonoid present in plants and berries that has been shown to limit cell growth and protect against cancer in in-vitro and animal models, was conducted using dictyostelium, a simple, controllable biomedical model. Cultured MDCK cells were used to generate differentiated tubule cells, and these findings were extrapolated to a human kidney model employing these cells. Results: While naringenin inhibited growth in dictyostelium, it had no effect on development. In a random-gene-knockout screen, a TRPP2 (polycystin-2) knockout mutant was discovered to be resistant to naringenin’s effects on growth and random-cell movement. Changes in the divalent transient receptor cause polycystic kidney disease type 2 potential cation channel TRPP2. We found that the growth of cysts and MDCK cells might be inhibited by naringenin. Partial resistance to naringenin was achieved in this model by lowering TRPP2 levels via siRNA, as evidenced by the presence of larger cysts following treatment with 3 and 10 M naringenin compared to controls. Naringenin had no effect on chloride secretion. Conclusion: Naringenin’s influence on cell proliferation is mediated by TRPP2 in both dictyostelium and mammalian kidney cells, despite their vast evolutionary distance from one another (polycystin-2). Naringenin will be the subject of more research as a possible new therapeutic treatment for ADPKD

Mathematical models of cystic fibrosis as a systemic disease.

Cystic fibrosis (CF) is widely known as a disease of the lung, even though it is in truth a systemic disease, whose symptoms typically manifest in gastrointestinal dysfunction first. CF ultimately impairs not only the pancreas and intestine but also the lungs, gonads, liver, kidneys, bones, and the cardiovascular system. It is caused by one of several mutations in the gene of the epithelial ion channel protein CFTR. Intense research and improved antimicrobial treatments during the past eight decades have steadily increased the predicted life expectancy of a person with CF (pwCF) from a few weeks to over 50 years. Moreover, several drugs ameliorating the sequelae of the disease have become available in recent years, and notable treatments of the root cause of the disease have recently generated substantial improvements in health for some but not all pwCF. Yet, numerous fundamental questions remain unanswered. Complicating CF, for instance in the lung, is the fact that the associated insufficient chloride secretion typically perturbs the electrochemical balance across epithelia and, in the airways, leads to the accumulation of thick, viscous mucus and mucus plaques that cannot be cleared effectively and provide a rich breeding ground for a spectrum of bacterial and fungal communities. The subsequent infections often become chronic and respond poorly to antibiotic treatments, with outcomes sometimes only weakly correlated with the drug susceptibility of the target pathogen. Furthermore, in contrast to rapidly resolved acute infections with a single target pathogen, chronic infections commonly involve multi-species bacterial communities, called "infection microbiomes," that develop their own ecological and evolutionary dynamics. It is presently impossible to devise mathematical models of CF in its entirety, but it is feasible to design models for many of the distinct drivers of the disease. Building upon these growing yet isolated modeling efforts, we discuss in the following the feasibility of a multi-scale modeling framework, known as template-and-anchor modeling, that allows the gradual integration of refined sub-models with different granularity. The article first reviews the most important biomedical aspects of CF and subsequently describes mathematical modeling approaches that already exist or have the potential to deepen our understanding of the multitude aspects of the disease and their interrelationships. The conceptual ideas behind the approaches proposed here do not only pertain to CF but are translatable to other systemic diseases. This article is categorized under: Congenital Diseases > Computational Models.

Reduced Plasma Guanylin Levels Following Enterotoxigenic Escherichia coli-Induced Diarrhea.

The intestinal peptide hormones guanylin (GN) and uroguanylin (UGN) interact with the epithelial cell receptor guanylate cyclase C to regulate fluid homeostasis. Some enterotoxigenic Escherichia coli (ETEC) produce heat-stable enterotoxin (ST), which induces diarrhea by mimicking GN and UGN. Plasma concentrations of prohormones of GN (proGN) and UGN (proUGN) are reportedly decreased during chronic diarrheal diseases. Here we investigate whether prohormone concentrations also drop during acute diarrhea caused by ST-producing ETEC strains TW10722 and TW11681. Twenty-one volunteers were experimentally infected with ETEC. Blood (n = 21) and urine (n = 9) specimens were obtained immediately before and 1, 2, 3, and 7 days after ETEC ingestion. Concentrations of proGN and proUGN were measured by ELISA. Urine electrolyte concentrations were measured by photometry and mass spectrometry. Ten volunteers developed diarrhea (D group), and eleven did not (ND group). In the D group, plasma proGN, but not proUGN, concentrations were substantially reduced on days 2 and 3, coinciding with one day after diarrhea onset. No changes were seen in the ND group. ETEC diarrhea also seemed to affect diuresis, the zinc/creatinine ratio, and sodium and chloride secretion levels in urine. ETEC-induced diarrhea causes a reduction in plasma proGN and could potentially be a useful marker for intestinal isotonic fluid loss.

The revolution of personalized pharmacotherapies for cystic fibrosis: what does the future hold?

ABSTRACT Introduction Cystic fibrosis (CF), a potentially fatal genetic disease, is caused by loss-of-function mutations in the gene encoding for the CFTR chloride/bicarbonate channel. Modulator drugs rescuing mutant CFTR traffic and function are now in the clinic, providing unprecedented breakthrough therapies for people with CF (PwCF) carrying specific genotypes. However, several CFTR variants are unresponsive to these therapies. Area covered We discussed several therapeutic approaches that are under development to tackle the fundamental cause of CF, including strategies targeting defective CFTR mRNA and/or protein expression and function. Alternatively, defective chloride secretion and dehydration in CF epithelia could be restored by exploiting pharmacological modulation of alternative targets, i.e., ion channels/transporters that concur with CFTR to maintain the airway surface liquid homeostasis (e.g., ENaC, TMEM16A, SLC26A4, SLC26A9, and ATP12A). Finally, we assessed progress and challenges in the development of gene-based therapies to replace or correct the mutant CFTR gene. Expert opinion CFTR modulators are benefiting many PwCF responsive to these drugs, yielding substantial improvements in various clinical outcomes. Meanwhile, the CF therapy development pipeline continues to expand with the development of novel CFTR modulators and alternative therapeutic strategies with the ultimate goal of providing effective therapies for all PwCF in the foreseeable future.

Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination.

Despite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination.