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Elias JE, Debela M, Sewell GW, Stopforth RJ, Partl H, Heissbauer S, Holland LM, Karlsen TH, Kaser A, Kaneider NC. GPR35 prevents osmotic stress induced cell damage. Commun Biol 2025; 8:478. [PMID: 40121360 PMCID: PMC11929815 DOI: 10.1038/s42003-025-07848-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2024] [Accepted: 02/28/2025] [Indexed: 03/25/2025] Open
Abstract
GPR35 is an orphan G-protein coupled receptor that has been implicated in the development of cancer. GPR35 regulates the Na+/K+-ATPase's pump and signalling function. Here we show GPR35's critical role in ion flux that in turn controls cellular osmotic pressure and Na+-dependent transport in HepG2 and SW480 cells. GPR35 deficiency results in increased levels of intracellular Na+, osmotic stress and changes in osmolytes leading to increased cells size and decreased glutamine import in vitro and in vivo. The GPR35-T108M risk variant, which increases risk for primary sclerosing cholangitis and inflammatory bowel disease, leads to lower intracellular Na+ levels, and enhanced glutamine uptake. High salt diet (HSD) in wildtype mice resembles the intestinal epithelial phenotype of their Gpr35-/- littermates with decreased Goblet cell size and numbers. This indicates that GPR35's regulation of the Na+/K+-ATPase controls ion homeostasis, osmosis and Na+-dependent transporters.
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Affiliation(s)
- Joshua E Elias
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Mekdes Debela
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK
| | - Gavin W Sewell
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Richard J Stopforth
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK
| | - Hannah Partl
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK
| | - Sophie Heissbauer
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK
| | - Lorraine M Holland
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK
| | - Tom H Karlsen
- Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway
| | - Arthur Kaser
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Nicole C Kaneider
- Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Cambridge, CB2 0AW, UK.
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge, CB2 0QQ, UK.
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Bayoumy AB, Derijks LJJ, Oldenburg B, de Boer NKH. The Use of Tissue Concentrations of Biological and Small-Molecule Therapies in Clinical Studies of Inflammatory Bowel Diseases. Pharmaceutics 2024; 16:1497. [PMID: 39771479 PMCID: PMC11676153 DOI: 10.3390/pharmaceutics16121497] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2024] [Revised: 11/15/2024] [Accepted: 11/19/2024] [Indexed: 01/11/2025] Open
Abstract
The introduction of biological therapies has revolutionized inflammatory bowel disease (IBD) management. A critical consideration in developing these therapies is ensuring adequate drug concentrations at the site of action. While blood-based biomarkers have shown limited utility in optimizing treatment (except for TNF-alpha inhibitors and thiopurines), tissue drug concentrations may offer valuable insights. In antimicrobial therapies, tissue concentration monitoring is standard practice and could provide a new avenue for understanding the pharmacokinetics of biological and small-molecule therapies in IBD. Various methods exist for measuring tissue concentrations, including whole tissue sampling, MALDI-MSI, microdialysis, and fluorescent labeling. These techniques offer unique advantages, such as spatial drug-distribution mapping, continuous sampling, or cellular-level analysis. However, challenges remain, including sampling invasiveness, heterogeneity in tissue compartments, and a lack of standardized bioanalytical guidelines. Drug pharmacokinetics are influenced by multiple factors, including molecular properties, disease-induced changes in the gastrointestinal tract, and the timing of sample collection. For example, drug permeability, solubility, and interaction with transporters may vary between Crohn's disease and ulcerative colitis. Research into the tissue concentrations of drugs like anti-TNF agents, ustekinumab, vedolizumab, and tofacitinib has shown variable correlations with clinical outcomes, suggesting potential roles for tissue concentration monitoring in therapeutic drug management. Although routine clinical application is not yet established, exploring tissue drug concentrations may enhance understanding of IBD pharmacotherapy.
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Affiliation(s)
- Ahmed B. Bayoumy
- Department of Gastroenterology and Hepatology, Amsterdam University Medical Center, 1105 AZ Amsterdam, The Netherlands;
- Amsterdam Gastroenterology Endocrinology Metabolism Research Institute, 1105 AZ Amsterdam, The Netherlands
| | - Luc J. J. Derijks
- Department of Clinical Pharmacy & Pharmacology, Máxima Medical Centre, 5631 BM Eindhoven, The Netherlands
- Department of Clinical Pharmacy & Toxicology and NUTRIM, School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre, 6229 HX Maastricht, The Netherlands
| | - Bas Oldenburg
- Department of Gastroenterology and Hepatology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands
| | - Nanne K. H. de Boer
- Department of Gastroenterology and Hepatology, Amsterdam University Medical Center, 1105 AZ Amsterdam, The Netherlands;
- Amsterdam Gastroenterology Endocrinology Metabolism Research Institute, 1105 AZ Amsterdam, The Netherlands
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3
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Feng G, Zhang H, Liu H, Zhang X, Jiang H, Liao S, Luo X, Yao H, Xiang B, Liu S, Zhang J, Zhang J, Fang J. Natural Flavonoid-Derived Enzyme Mimics DHKNase Balance the Two-Edged Reactive Oxygen Species Function for Wound Healing and Inflammatory Bowel Disease Therapy. RESEARCH (WASHINGTON, D.C.) 2024; 7:0464. [PMID: 39253100 PMCID: PMC11381673 DOI: 10.34133/research.0464] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 06/13/2024] [Accepted: 08/07/2024] [Indexed: 09/11/2024]
Abstract
Rational regulation of reactive oxygen species (ROS) plays a vital importance in maintaining homeostasis of living biological systems. For ROS-related pathologies, chemotherapy technology derived from metal nanomaterials currently occupies a pivotal position. However, they suffer from inherent issues such as complicated synthesis, batch-to-batch variability, high cost, and potential biological toxicity caused by metal elements. Here, we reported for the first time that dual-action 3,5-dihydroxy-1-ketonaphthalene-structured small-molecule enzyme imitator (DHKNase) exhibited 2-edged ROS regulation, catering to the execution of physiology-beneficial ROS destiny among diverse pathologies in living systems. Based on this, DHKNase is validated to enable remarkable therapeutic effects in 2 classic disease models, including the pathogen-infected wound-healing model and the dextran sulfate sodium (DSS)-caused inflammatory bowel disease (IBD). This work provides a guiding landmark for developing novel natural small-molecule enzyme imitator and significantly expands their application potential in the biomedical field.
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Affiliation(s)
- Guangfu Feng
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
| | - Huaizu Zhang
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
| | - Huipeng Liu
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
| | - Xiaoyan Zhang
- College of Life Science, Shihezi University, Shihezi, Xinjiang 832003, P.R. China
| | - Hongmei Jiang
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
| | - Sijie Liao
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
| | - Xingyu Luo
- School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, P.R. China
| | - Hao Yao
- Changsha IMADEK Intelligent Technology Co. Ltd., Changsha, Hunan 410081, P.R. China
| | - Bo Xiang
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
| | - Shiyu Liu
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
| | - Jiali Zhang
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
| | - Jiaheng Zhang
- College of Chemistry, Food Laboratory of Zhongyuan, Flavour Science Research Center of Zhengzhou University, Zhengzhou University, Zhengzhou, Henan 450001, P.R. China
| | - Jun Fang
- School of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P.R. China
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Tang LQ, Fraebel J, Jin S, Winesett SP, Harrell J, Chang WH, Cheng SX. Calcium/calcimimetic via calcium-sensing receptor ameliorates cholera toxin-induced secretory diarrhea in mice. World J Gastroenterol 2024; 30:268-279. [PMID: 38314127 PMCID: PMC10835527 DOI: 10.3748/wjg.v30.i3.268] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/02/2023] [Revised: 12/01/2023] [Accepted: 01/02/2024] [Indexed: 01/18/2024] Open
Abstract
BACKGROUND Enterotoxins produce diarrhea through direct epithelial action and indirectly by activating the enteric nervous system. Calcium-sensing receptor (CaSR) inhibits both actions. The latter has been well documented in vitro but not in vivo. The hypothesis to be tested was that activating CaSR inhibits diarrhea in vivo. AIM To determine whether CaSR agonists ameliorate secretory diarrhea evoked by cholera toxin (CTX) in mice. METHODS CTX was given orally to C57BL/6 mice to induce diarrhea. Calcium and calcimimetic R568 were used to activate CaSR. To maximize their local intestinal actions, calcium was administered luminally via oral rehydration solution (ORS), whereas R568 was applied serosally using an intraperitoneal route. To verify that their actions resulted from the intestine, effects were also examined on Cre-lox intestine-specific CaSR knockouts. Diarrhea outcome was measured biochemically by monitoring changes in fecal Cl- or clinically by assessing stool consistency and weight loss. RESULTS CTX induced secretory diarrhea, as evidenced by increases in fecal Cl-, stool consistency, and weight loss following CTX exposure, but did not alter CaSR, neither in content nor in function. Accordingly, calcium and R568 were each able to ameliorate diarrhea when applied to diseased intestines. Intestinal CaSR involvement is suggested by gene knockout experiments where the anti-diarrheal actions of R568 were lost in intestinal epithelial CaSR knockouts (villinCre/Casrflox/flox) and neuronal CaSR knockouts (nestinCre/Casrflox/flox). CONCLUSION Treatment of acute secretory diarrheas remains a global challenge. Despite advances in diarrhea research, few have been made in the realm of diarrhea therapeutics. ORS therapy has remained the standard of care, although it does not halt the losses of intestinal fluid and ions caused by pathogens. There is no cost-effective therapeutic for diarrhea. This and other studies suggest that adding calcium to ORS or using calcimimetics to activate intestinal CaSR might represent a novel approach for treating secretory diarrheal diseases.
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Affiliation(s)
- Lie-Qi Tang
- Department of Pediatrics, University of Florida, Gainesville, FL 32610, United States
| | - Johnathan Fraebel
- Department of Pediatrics, University of Florida, Gainesville, FL 32610, United States
- College of Medicine, University of Florida, Gainesville, FL 32610, United States
| | - Shi Jin
- Department of Pediatrics, University of Florida, Gainesville, FL 32610, United States
| | - Steven P Winesett
- Applied Physiology and Kinesiology, University of Florida, Gainesville, FL 32610, United States
- Brain Rehabilitation Research Center, Malcom Randall VA Medical Center, Gainesville, FL 32610, United States
| | - Jane Harrell
- Department of Pediatrics, University of Florida, Gainesville, FL 32610, United States
| | - Wen-Han Chang
- Department of Medicine, Endocrine Research Unit, Veterans Affairs Medical Center, University of California, San Francisco, San Francisco, CA 94121, United States
| | - Sam Xianjun Cheng
- Department of Pediatric Gastroenterology, Hepatology, and Nutrition, University of Florida Shands Children’s Hospital, Gainesville, FL 32608, United States
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Stuard Sambhariya W, Trautmann IJ, Robertson DM. Insulin-like growth factor binding protein-3 mediates hyperosmolar stress-induced mitophagy through the mechanistic target of rapamycin. J Biol Chem 2023; 299:105239. [PMID: 37690686 PMCID: PMC10637961 DOI: 10.1016/j.jbc.2023.105239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2022] [Revised: 07/29/2023] [Accepted: 08/04/2023] [Indexed: 09/12/2023] Open
Abstract
Hyperosmolarity of the ocular surface triggers inflammation and pathological damage in dry eye disease (DED). In addition to a reduction in quality of life, DED causes vision loss and when severe, blindness. Mitochondrial dysfunction occurs as a consequence of hyperosmolar stress. We have previously reported on a role for the insulin-like growth factor binding protein-3 (IGFBP-3) in the regulation of mitochondrial ultrastructure and metabolism in mucosal surface epithelial cells; however, this appears to be context-specific. Due to the finding that IGFBP-3 expression is decreased in response to hyperosmolar stress in vitro and in an animal model of DED, we next sought to determine whether the hyperosmolar stress-mediated decrease in IGFBP-3 alters mitophagy, a key mitochondrial quality control mechanism. Here we show that hyperosmolar stress induces mitophagy through differential regulation of BNIP3L/NIX and PINK1-mediated pathways. In corneal epithelial cells, this was independent of p62. The addition of exogenous IGFBP-3 abrogated the increase in mitophagy. This occurred through regulation of mTOR, highlighting the existence of a new IGFBP-3-mTOR signaling pathway. Together, these findings support a novel role for IGFBP-3 in mediating mitochondrial quality control in DED and have broad implications for epithelial tissues subject to hyperosmolar stress and other mitochondrial diseases.
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Affiliation(s)
- Whitney Stuard Sambhariya
- Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Ian J Trautmann
- Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Danielle M Robertson
- Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
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Sänger CS, Cernakova M, Wietecha MS, Garau Paganella L, Labouesse C, Dudaryeva OY, Roubaty C, Stumpe M, Mazza E, Tibbitt MW, Dengjel J, Werner S. Serine protease 35 regulates the fibroblast matrisome in response to hyperosmotic stress. SCIENCE ADVANCES 2023; 9:eadh9219. [PMID: 37647410 PMCID: PMC10468140 DOI: 10.1126/sciadv.adh9219] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Accepted: 07/28/2023] [Indexed: 09/01/2023]
Abstract
Hyperosmotic stress occurs in several diseases, but its long-term effects are largely unknown. We used sorbitol-treated human fibroblasts in 3D culture to study the consequences of hyperosmotic stress in the skin. Sorbitol regulated many genes, which help cells cope with the stress condition. The most robustly regulated gene encodes serine protease 35 (PRSS35). Its regulation by hyperosmotic stress was dependent on the kinases p38 and JNK and the transcription factors NFAT5 and ATF2. We identified different collagens and collagen-associated proteins as putative PRSS35 binding partners. This is functionally important because PRSS35 affected the extracellular matrix proteome, which limited cell proliferation. The in vivo relevance of these findings is reflected by the coexpression of PRSS35 and its binding partners in human skin wounds, where hyperosmotic stress occurs as a consequence of excessive water loss. These results identify PRSS35 as a key regulator of the matrisome under hyperosmotic stress conditions.
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Affiliation(s)
- Catharina S. Sänger
- Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Otto-Stern-Weg 7, 8093 Zurich, Switzerland
| | - Martina Cernakova
- Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Otto-Stern-Weg 7, 8093 Zurich, Switzerland
| | - Mateusz S. Wietecha
- Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Otto-Stern-Weg 7, 8093 Zurich, Switzerland
- Department of Oral Biology, College of Dentistry, University of Illinois Chicago, Chicago, IL 60612, USA
| | - Lorenza Garau Paganella
- Institute for Mechanical Systems, Department of Mechanical and Process Engineering, ETH Zurich, Leonhardstrasse 21, 8092 Zurich, Switzerland
- Institute for Energy and Process Engineering, Department of Mechanical and Process Engineering, ETH Zurich, Sonneggstrasse 3, 8092 Zurich, Switzerland
| | - Céline Labouesse
- Institute for Energy and Process Engineering, Department of Mechanical and Process Engineering, ETH Zurich, Sonneggstrasse 3, 8092 Zurich, Switzerland
| | - Oksana Y. Dudaryeva
- Institute for Energy and Process Engineering, Department of Mechanical and Process Engineering, ETH Zurich, Sonneggstrasse 3, 8092 Zurich, Switzerland
| | - Carole Roubaty
- Faculty of Science and Medicine, Department of Biology, University of Fribourg, Ch. du Musée 10, 1700 Fribourg, Switzerland
| | - Michael Stumpe
- Faculty of Science and Medicine, Department of Biology, University of Fribourg, Ch. du Musée 10, 1700 Fribourg, Switzerland
| | - Edoardo Mazza
- Institute for Mechanical Systems, Department of Mechanical and Process Engineering, ETH Zurich, Leonhardstrasse 21, 8092 Zurich, Switzerland
| | - Mark W. Tibbitt
- Institute for Energy and Process Engineering, Department of Mechanical and Process Engineering, ETH Zurich, Sonneggstrasse 3, 8092 Zurich, Switzerland
| | - Jörn Dengjel
- Faculty of Science and Medicine, Department of Biology, University of Fribourg, Ch. du Musée 10, 1700 Fribourg, Switzerland
| | - Sabine Werner
- Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Otto-Stern-Weg 7, 8093 Zurich, Switzerland
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Nighot M, Liao PL, Morris N, McCarthy D, Dharmaprakash V, Ullah Khan I, Dalessio S, Saha K, Ganapathy AS, Wang A, Ding W, Yochum G, Koltun W, Nighot P, Ma T. Long-Term Use of Proton Pump Inhibitors Disrupts Intestinal Tight Junction Barrier and Exaggerates Experimental Colitis. J Crohns Colitis 2023; 17:565-579. [PMID: 36322638 PMCID: PMC10115233 DOI: 10.1093/ecco-jcc/jjac168] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2022] [Indexed: 12/03/2022]
Abstract
BACKGROUND Proton pump inhibitors [PPIs] are widely used to treat a number of gastro-oesophageal disorders. PPI-induced elevation in intragastric pH may alter gastrointestinal physiology. The tight junctions [TJs] residing at the apical intercellular contacts act as a paracellular barrier. TJ barrier dysfunction is an important pathogenic factor in inflammatory bowel disease [IBD]. Recent studies suggest that PPIs may promote disease flares in IBD patients. The role of PPIs in intestinal permeability is not clear. AIM The aim of the present study was to study the effect of PPIs on the intestinal TJ barrier function. METHODS Human intestinal epithelial cell culture and organoid models and mouse IBD models of dextran sodium sulphate [DSS] and spontaneous enterocolitis in IL-10-/- mice were used to study the role of PPIs in intestinal permeability. RESULTS PPIs increased TJ barrier permeability via an increase in a principal TJ regulator, myosin light chain kinase [MLCK] activity and expression, in a p38 MAPK-dependent manner. The PPI-induced increase in extracellular pH caused MLCK activation via p38 MAPK. Long-term PPI administration in mice exaggerated the increase in intestinal TJ permeability and disease severity in two independent models of DSS colitis and IL-10-/- enterocolitis. The TJ barrier disruption by PPIs was prevented in MLCK-/- mice. Human database studies revealed increased hospitalizations associated with PPI use in IBD patients. CONCLUSIONS Our results suggest that long-term use of PPIs increases intestinal TJ permeability and exaggerates experimental colitis via an increase in MLCK expression and activity.
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Affiliation(s)
- Meghali Nighot
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | - Pei-Luan Liao
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | - Nathan Morris
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | - Dennis McCarthy
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of New Mexico, Albuquerque, NM 87131, USA
| | - Viszwapriya Dharmaprakash
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | - Inam Ullah Khan
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | - Shannon Dalessio
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | - Kushal Saha
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | | | - Alexandra Wang
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | - Wei Ding
- Division of Colon and Rectal Surgery, Department of Surgery, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
| | - Gregory Yochum
- Division of Colon and Rectal Surgery, Department of Surgery, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
| | - Walter Koltun
- Division of Colon and Rectal Surgery, Department of Surgery, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
| | - Prashant Nighot
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
| | - Thomas Ma
- Division of Gastroenterology and Hepatology, Department of Medicine, Pennsylvania State College of Medicine, Hershey, PA 17033, USA
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Cao D, Wang S, Zhang D, Zhang Y, Cao J, Liu Y, Zhou H. KRAB family is involved in network shifts in response to osmotic stress in camels. Anim Cells Syst (Seoul) 2022; 26:348-357. [PMID: 36605583 PMCID: PMC9809417 DOI: 10.1080/19768354.2022.2143894] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
A feature of the camel is its tolerance to osmotic stress. However, few studies of osmotic stress in vivo or comparative analyses between different tissues of the camel have been performed. Here, we report the roles of Krüppel-associated box domain containing zinc-finger repressor proteins (KRAB-ZFPs) in transcriptional networks under osmotic stress in camels by analyzing transcriptomes of four different tissues under various osmotic conditions. We found that 273 of 278 KRAB-ZFPs were expressed in our data set, being involved in all of the 65 identified networks and exhibiting their extensive functional diversity. We also found that 110 KRAB-ZFPs were hub genes involved in more than half of the networks. We demonstrated that the osmotic stress response is involved in network shifts and that KRAB-ZFPs mediate this process. Finally, we presented the diverse mechanisms of osmotic stress responses in different tissues. These results revealed the genetic architecture of systematic physiological response in vivo to osmotic stress in camels. Our work will lead to new directions for studying the mechanism of osmotic stress response in anti-arid mammals.
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Affiliation(s)
- Dandan Cao
- College of Life Science, Inner Mongolia Agricultural University, Hohhot, People’s Republic of China
| | - Shenyuan Wang
- College of Life Science, Inner Mongolia Agricultural University, Hohhot, People’s Republic of China,Sheep Collaboration and Innovation Center, Inner Mongolia University, Hohhot, People’s Republic of China
| | - Dong Zhang
- College of Life Science, Inner Mongolia Agricultural University, Hohhot, People’s Republic of China
| | - Yanru Zhang
- College of Life Science, Inner Mongolia Agricultural University, Hohhot, People’s Republic of China
| | - Junwei Cao
- College of Life Science, Inner Mongolia Agricultural University, Hohhot, People’s Republic of China
| | - Yongbin Liu
- Sheep Collaboration and Innovation Center, Inner Mongolia University, Hohhot, People’s Republic of China
| | - Huanmin Zhou
- College of Life Science, Inner Mongolia Agricultural University, Hohhot, People’s Republic of China,Sheep Collaboration and Innovation Center, Inner Mongolia University, Hohhot, People’s Republic of China, Huanmin Zhou College of Life Science, Inner Mongolia Agricultural University, No. 306 Zhaowuda Road, Hohhot, Inner Mongolia010018, People’s Republic of China; Sheep Collaboration and Innovation Center, Inner Mongolia University, No. 235 Daxue West Street, Hohhot, Inner Mongolia010021, People’s Republic of China
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9
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Hypo-osmotic stress induces the epithelial alarmin IL-33 in the colonic barrier of ulcerative colitis. Sci Rep 2022; 12:11550. [PMID: 35798804 PMCID: PMC9263100 DOI: 10.1038/s41598-022-15573-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2022] [Accepted: 06/27/2022] [Indexed: 11/08/2022] Open
Abstract
Epithelial alarmins are gaining interest as therapeutic targets for chronic inflammation. The nuclear alarmin interleukin-33 (IL-33) is upregulated in the colonic mucosa of acute ulcerative colitis (UC) and may represent an early instigator of the inflammatory cascade. However, it is not clear what signals drive the expression of IL-33 in the colonic mucosa, nor is the exact role of IL-33 elucidated. We established an ex vivo model using endoscopic colonic biopsies from healthy controls and UC patients. Colonic biopsies exposed to hypo-osmotic medium induced a strong nuclear IL-33 expression in colonic crypts in both healthy controls and UC biopsies. Mucosal IL33 mRNA was also significantly increased following hypo-osmotic stress in healthy controls compared to non-stimulated biopsies (fold change 3.9, p-value < 0.02). We observed a modest induction of IL-33 in response to TGF-beta-1 stimulation, whereas responsiveness to inflammatory cytokines TNF and IFN-gamma was negligible. In conclusion our findings indicate that epithelial IL-33 is induced by hypo-osmotic stress, rather than prototypic proinflammatory cytokines in colonic ex vivo biopsies. This is a novel finding, linking a potent cytokine and alarmin of the innate immune system with cellular stress mechanisms and mucosal inflammation.
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10
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Lactobacillus casei and Epidermal Growth Factor Prevent Osmotic Stress-Induced Tight Junction Disruption in Caco-2 Cell Monolayers. Cells 2021; 10:cells10123578. [PMID: 34944085 PMCID: PMC8700399 DOI: 10.3390/cells10123578] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2021] [Revised: 12/13/2021] [Accepted: 12/15/2021] [Indexed: 01/08/2023] Open
Abstract
Osmotic stress plays a crucial role in the pathogenesis of many gastrointestinal diseases. Lactobacillus casei and epidermal growth factor (EGF) effects on the osmotic stress-induced epithelial junctional disruption and barrier dysfunction were investigated. Caco-2 cell monolayers were exposed to osmotic stress in the presence or absence of L. casei or EGF, and the barrier function was evaluated by measuring inulin permeability. Tight junction (TJ) and adherens junction integrity were assessed by immunofluorescence confocal microscopy. The role of signaling molecules in the L. casei and EGF effects was determined by using selective inhibitors. Data show that pretreatment of cell monolayers with L. casei or EGF attenuates osmotic stress-induced TJ and adherens junction disruption and barrier dysfunction. EGF also blocked osmotic stress-induced actin cytoskeleton remodeling. U0126 (MEK1/2 inhibitor), the MAP kinase inhibitor, blocked EGF-mediated epithelial protection from osmotic stress. In contrast, the L. casei-mediated epithelial protection from osmotic stress was unaffected by U0126, AG1478 (EGFR tyrosine kinase inhibitor), SP600125 (JNK1/2 inhibitor), or SB202190 (P38 MAP kinase inhibitor). On the other hand, Ro-32-0432 (PKC inhibitor) blocked the L. casei-mediated prevention of osmotic stress-induced TJ disruption and barrier dysfunction. The combination of EGF and L. casei is more potent in protecting the barrier function from osmotic stress. These findings suggest that L. casei and EGF ameliorate osmotic stress-induced disruption of apical junctional complexes and barrier dysfunction in the intestinal epithelium by distinct signaling mechanisms.
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11
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Hyperosmolarity Triggers the Warburg Effect in Chinese Hamster Ovary Cells and Reveals a Reduced Mitochondria Horsepower. Metabolites 2021; 11:metabo11060344. [PMID: 34073567 PMCID: PMC8226498 DOI: 10.3390/metabo11060344] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2021] [Accepted: 05/18/2021] [Indexed: 12/22/2022] Open
Abstract
Tumor cells are known to favor a glycolytic metabolism over oxidative phosphorylation (OxPhos), which takes place in mitochondria, to produce the energy and building blocks essential for cell maintenance and cell growth. This phenotypic property of tumor cells gives them several advantages over normal cells and is known as the Warburg effect. Tumors can be treated as a metabolic disease by targeting their bioenergetics capacity. Alpha-lipoic acid (ALA) and calcium hydroxycitrate (HCA) are two drugs known to target the Warburg effect in tumor cells and hence induce the mitochondria for ATP production. However, tumor cells, known to have an increased flux through glycolysis, are not able to handle the activation of their mitochondria by drugs or any other condition, leading to decoupling of gene regulation. In this study, these drug effects were studied by mimicking an inflammatory condition through the imposition of a hyperosmotic condition in Chinese hamster ovary (CHO) cells, which behave similarly to tumor cells. Indeed, CHO cells grown in high osmolarity conditions, using 200 mM mannitol, showed a pronounced Warburg effect phenotype. Our results show that hyperosmolar conditions triggered high-throughput glycolysis and enhanced glutaminolysis in CHO cells, such as during cancer cell proliferation in inflammatory tissue. Finally, we found that the hyperosmolar condition was correlated with increased mitochondrial membrane potential (ΔΨm) but mitochondrial horsepower seemed to vanish (h = Δp/ΔΨm), which may be explained by mitochondrial hyperfusion.
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12
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Effinger A, O'Driscoll CM, McAllister M, Fotaki N. Predicting budesonide performance in healthy subjects and patients with Crohn's disease using biorelevant in vitro dissolution testing and PBPK modeling. Eur J Pharm Sci 2021; 157:105617. [PMID: 33164838 DOI: 10.1016/j.ejps.2020.105617] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2020] [Revised: 10/02/2020] [Accepted: 10/19/2020] [Indexed: 02/07/2023]
Abstract
OBJECTIVES Drug product performance might be affected in Crohn's disease (CD) patients compared to healthy subjects due to pathophysiological changes. Since a low number of clinical studies is performed in this patient population, physiologically-based pharmacokinetic (PBPK) models with integrated results from biorelevant in vitro dissolution studies could be used to assess differences in the bioavailability of drugs. Using this approach, budesonide was used as model drug and its performance in healthy subjects and CD patients was predicted and compared against observed pharmacokinetic data. The in vitro release tests, under healthy versus CD conditions, revealed a similar extent of drug release from a controlled-release budesonide formulation in the fasted state, whereas in the fed state a lower extent was observed with CD. Differences in the physiology of CD patients were identified in literature and their impact on budesonide performance was investigated with a PBPK model, revealing the highest impact on the simulated bioavailability for the reduced hepatic CYP3A4 enzyme abundance and lower human serum albumin concentration. For CD patients, a higher budesonide exposure compared to healthy subjects was predicted with a PBPK population adapted to CD physiology and in agreement with observed pharmacokinetic data. Budesonide performance in the fasted and fed state was successfully predicted in healthy subjects and CD patients using PBPK modeling and in vitro release testing. Following this approach, predictions of the direction and magnitude of changes in bioavailability due to CD could be made for other drugs and guide prescribers to adjust dosage regimens for CD patients accordingly.
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Affiliation(s)
- Angela Effinger
- Department of Pharmacy and Pharmacology, University of Bath, Bath, UK
| | | | | | - Nikoletta Fotaki
- Department of Pharmacy and Pharmacology, University of Bath, Bath, UK.
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13
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Gastrointestinal diseases and their impact on drug solubility: Crohn's disease. Eur J Pharm Sci 2020; 152:105459. [PMID: 32649984 DOI: 10.1016/j.ejps.2020.105459] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Revised: 06/12/2020] [Accepted: 07/05/2020] [Indexed: 12/12/2022]
Abstract
In order to investigate differences in drug solubilisation and dissolution in luminal fluids of Crohn's disease (CD) patients and healthy subjects, biorelevant media representative of CD patients were developed using information from literature and a Design of Experiment (DoE) approach. The CD media were characterised in terms of surface tension, osmolality, dynamic viscosity and buffer capacity and compared to healthy biorelevant media. To identify which drug characteristics are likely to present a high risk of altered drug solubility in CD, the solubility of six drugs was assessed in CD media and solubility differences were related to drug properties. Identified differences in CD patients compared to healthy subjects were a reduced concentration of bile salts, a higher gastric pH and a higher colonic osmolality. Differences in the properties of CD compared to healthy biorelevant media were mainly observed for surface tension and osmolality. Drug solubility of ionisable compounds was altered in gastric CD media compared to healthy biorelevant media. For drugs with moderate to high lipophilicity, a high risk of altered drug solubilisation in CD is expected, since a significant negative effect of log P and a positive effect of bile salts on drug solubility in colonic and fasted state intestinal CD media was observed. Simulating the conditions in CD patients in vitro offers the possibility to identify relevant differences in drug solubilisation without conducting expensive clinical trials.
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14
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Inflammatory bowel disease-associated ubiquitin ligase RNF183 promotes lysosomal degradation of DR5 and TRAIL-induced caspase activation. Sci Rep 2019; 9:20301. [PMID: 31889078 PMCID: PMC6937276 DOI: 10.1038/s41598-019-56748-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Accepted: 12/16/2019] [Indexed: 02/07/2023] Open
Abstract
RNF183 is a ubiquitin ligase containing RING-finger and transmembrane domains, and its expression levels are increased in patients with inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, and in 2,4,6-trinitrobenzene sulfonic acid-induced colitis mice. Here, we further demonstrate that RNF183 was induced to a greater degree in the dextran sulfate sodium (DSS)-treated IBD model at a very early stage than were inflammatory cytokines. In addition, fluorescence-activated cell sorting and polymerase chain reaction analysis revealed that RNF183 was specifically expressed in epithelial cells of DSS-treated mice, which suggested that increased levels of RNF183 do not result from the accumulation of immune cells. Furthermore, we identified death receptor 5 (DR5), a member of tumour necrosis factor (TNF)-receptor superfamily, as a substrate of RNF183. RNF183 mediated K63-linked ubiquitination and lysosomal degradation of DR5. DR5 promotes TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis signal through interaction with caspase-8. Inhibition of RNF183 expression was found to suppress TRAIL-induced activation of caspase-8 and caspase-3. Thus, RNF183 promoted not only DR5 transport to lysosomes but also TRAIL-induced caspase activation and apoptosis. Together, our results provide new insights into potential roles of RNF183 in DR5-mediated caspase activation in IBD pathogenesis.
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15
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Hamraz M, Abolhassani R, Andriamihaja M, Ransy C, Lenoir V, Schwartz L, Bouillaud F. Hypertonic external medium represses cellular respiration and promotes Warburg/Crabtree effect. FASEB J 2019; 34:222-236. [PMID: 31914644 DOI: 10.1096/fj.201900706rr] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2019] [Revised: 10/04/2019] [Accepted: 10/15/2019] [Indexed: 01/03/2023]
Abstract
Hyperosmotic conditions are associated to several pathological states. In this article, we evaluate the consequence of hyperosmotic medium on cellular energy metabolism. We demonstrate that exposure of cells to hyperosmotic conditions immediately reduces the mitochondrial oxidative phosphorylation rate. This causes an increase in glycolysis, which represses further respiration. This is known as the Warburg or Crabtree effect. In addition to aerobic glycolysis, we observed two other cellular responses that would help to preserve cellular ATP level and viability: A reduction in the cellular ATP turnover rate and a partial mitochondrial uncoupling which is expected to enhance ATP production by Krebs cycle. The latter is likely to constitute another metabolic adaptation to compensate for deficient oxidative phosphorylation that, importantly, is not dependent on glucose.
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Affiliation(s)
- Minoo Hamraz
- Institut Cochin, INSERM, CNRS, Université de Paris, Paris, France
| | | | - Mireille Andriamihaja
- INRA/AgroParisTech UMR 914, Physiologie de la Nutrition et du Comportement Alimentaire, Paris, France
| | - Céline Ransy
- Institut Cochin, INSERM, CNRS, Université de Paris, Paris, France
| | - Véronique Lenoir
- Institut Cochin, INSERM, CNRS, Université de Paris, Paris, France
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16
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Grauso M, Lan A, Andriamihaja M, Bouillaud F, Blachier F. Hyperosmolar environment and intestinal epithelial cells: impact on mitochondrial oxygen consumption, proliferation, and barrier function in vitro. Sci Rep 2019; 9:11360. [PMID: 31388052 PMCID: PMC6684637 DOI: 10.1038/s41598-019-47851-9] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2019] [Accepted: 07/22/2019] [Indexed: 01/01/2023] Open
Abstract
The aim of the present study was to elucidate the in vitro short-term (2-h) and longer-term (24-h) effects of hyperosmolar media (500 and 680 mOsm/L) on intestinal epithelial cells using the human colonocyte Caco-2 cell line model. We found that a hyperosmolar environment slowed down cell proliferation compared to normal osmolarity (336 mOsm/L) without inducing cell detachment or necrosis. This was associated with a transient reduction of cell mitochondrial oxygen consumption, increase in proton leak, and decrease in intracellular ATP content. The barrier function of Caco-2 monolayers was also transiently affected since increased paracellular apical-to-basal permeability and modified electrolyte permeability were measured, allowing partial equilibration of the trans-epithelial osmotic difference. In addition, hyperosmotic stress induced secretion of the pro-inflammatory cytokine IL-8. By measuring expression of genes involved in energy metabolism, tight junction forming, electrolyte permeability and intracellular signaling, different response patterns to hyperosmotic stress occurred depending on its intensity and duration. These data highlight the potential impact of increased luminal osmolarity on the intestinal epithelium renewal and barrier function and point out some cellular adaptive capacities towards luminal hyperosmolar environment.
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Affiliation(s)
- Marta Grauso
- UMR PNCA, AgroParisTech, INRA, Université Paris-Saclay, 75005, Paris, France.
| | - Annaïg Lan
- UMR PNCA, AgroParisTech, INRA, Université Paris-Saclay, 75005, Paris, France
| | | | - Frédéric Bouillaud
- Institut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, 75014, Paris, France
| | - François Blachier
- UMR PNCA, AgroParisTech, INRA, Université Paris-Saclay, 75005, Paris, France
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17
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Rasmussen RN, Christensen KV, Holm R, Nielsen CU. Transcriptome analysis identifies activated signaling pathways and regulated ABC transporters and solute carriers after hyperosmotic stress in renal MDCK I cells. Genomics 2018; 111:1557-1565. [PMID: 30389539 DOI: 10.1016/j.ygeno.2018.10.014] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2018] [Revised: 10/22/2018] [Accepted: 10/29/2018] [Indexed: 12/01/2022]
Abstract
Hyperosmolality is found under physiological conditions in the kidneys, whereas hyperosmolality in other tissues may be associated with pathological conditions. In such tissues an association between inflammation and hyperosmolality has been suggested. During hyperosmotic stress, an important phenomenon is upregulation of solute carriers (SLCs). We hypothesize that hyperosmolality affects the expression of many SLCs as well as ABC transporters. Through RNA-sequencing and topological pathway analysis, the cell cycle, the cytokine-cytokine receptor interaction pathway, and the chemokine-signaling pathway were significantly activated in MDCK I cells after hyperosmotic treatment (Δ200 mOsm) with raffinose or NaCl. 9065, 8052 and 5018 genes were significantly regulated by raffinose, NaCl or urea supplementation (500 mOsm), respectively, compared to control (300 mOsm). Cytokines, that have not previously been associated with hyperosmolality, were identified. We further provide an overview of transport proteins that could be of relevance in tissues exposed to hyperosmolality. Especially Slc5a8 was found highly up-regulated.
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Affiliation(s)
- Rune Nørgaard Rasmussen
- Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark..
| | | | - René Holm
- Drug Product Development, Janssens Research and Development, Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Belgium
| | - Carsten Uhd Nielsen
- Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
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18
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Ahmadi S, Xia S, Wu YS, Di Paola M, Kissoon R, Luk C, Lin F, Du K, Rommens J, Bear CE. SLC6A14, an amino acid transporter, modifies the primary CF defect in fluid secretion. eLife 2018; 7:37963. [PMID: 30004386 PMCID: PMC6054531 DOI: 10.7554/elife.37963] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 07/12/2018] [Indexed: 01/29/2023] Open
Abstract
The severity of intestinal disease associated with Cystic Fibrosis (CF) is variable in the patient population and this variability is partially conferred by the influence of modifier genes. Genome-wide association studies have identified SLC6A14, an electrogenic amino acid transporter, as a genetic modifier of CF-associated meconium ileus. The purpose of the current work was to determine the biological role of Slc6a14, by disrupting its expression in CF mice bearing the major mutation, F508del. We found that disruption of Slc6a14 worsened the intestinal fluid secretion defect, characteristic of these mice. In vitro studies of mouse intestinal organoids revealed that exacerbation of the primary defect was associated with reduced arginine uptake across the apical membrane, with aberrant nitric oxide and cyclic GMP-mediated regulation of the major CF-causing mutant protein. Together, these studies highlight the role of this apical transporter in modifying cellular nitric oxide levels, residual function of the major CF mutant and potentially, its promise as a therapeutic target.
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Affiliation(s)
- Saumel Ahmadi
- Department of Physiology, University of Toronto, Toronto, Canada.,Programme in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Sunny Xia
- Department of Physiology, University of Toronto, Toronto, Canada.,Programme in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Yu-Sheng Wu
- Department of Physiology, University of Toronto, Toronto, Canada.,Programme in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Michelle Di Paola
- Programme in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Canada.,Department of Biochemistry, University of Toronto, Toronto, Canada
| | - Randolph Kissoon
- Programme in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Catherine Luk
- Programme in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Fan Lin
- Department of Molecular Genetics, University of Toronto, Toronto, Canada
| | - Kai Du
- Programme in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Johanna Rommens
- Department of Molecular Genetics, University of Toronto, Toronto, Canada.,Programme in Genetics and Genome Biology, Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Christine E Bear
- Department of Physiology, University of Toronto, Toronto, Canada.,Programme in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, Canada.,Department of Biochemistry, University of Toronto, Toronto, Canada
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19
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Effinger A, O'Driscoll CM, McAllister M, Fotaki N. Impact of gastrointestinal disease states on oral drug absorption – implications for formulation design – a PEARRL review. J Pharm Pharmacol 2018; 71:674-698. [PMID: 29766501 DOI: 10.1111/jphp.12928] [Citation(s) in RCA: 58] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2017] [Accepted: 04/16/2018] [Indexed: 12/18/2022]
Abstract
Abstract
Objectives
Drug product performance in patients with gastrointestinal (GI) diseases can be altered compared to healthy subjects due to pathophysiological changes. In this review, relevant differences in patients with inflammatory bowel diseases, coeliac disease, irritable bowel syndrome and short bowel syndrome are discussed and possible in vitro and in silico tools to predict drug product performance in this patient population are assessed.
Key findings
Drug product performance was altered in patients with GI diseases compared to healthy subjects, as assessed in a limited number of studies for some drugs. Underlying causes can be observed pathophysiological alterations such as the differences in GI transit time, the composition of the GI fluids and GI permeability. Additionally, alterations in the abundance of metabolising enzymes and transporter systems were observed. The effect of the GI diseases on each parameter is not always evident as it may depend on the location and the state of the disease. The impact of the pathophysiological change on drug bioavailability depends on the physicochemical characteristics of the drug, the pharmaceutical formulation and drug metabolism. In vitro and in silico methods to predict drug product performance in patients with GI diseases are currently limited but could be a useful tool to improve drug therapy.
Summary
Development of suitable in vitro dissolution and in silico models for patients with GI diseases can improve their drug therapy. The likeliness of the models to provide accurate predictions depends on the knowledge of pathophysiological alterations, and thus, further assessment of physiological differences is essential.
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Affiliation(s)
- Angela Effinger
- Department of Pharmacy and Pharmacology, University of Bath, Bath, UK
| | | | | | - Nikoletta Fotaki
- Department of Pharmacy and Pharmacology, University of Bath, Bath, UK
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20
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Abstract
Inflammatory bowel diseases broadly categorized into Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders of the gastrointestinal tract with increasing prevalence worldwide. The etiology of the disease is complex and involves a combination of genetic, environmental, immunological and gut microbial factors. Recurring and bloody diarrhea is the most prevalent and debilitating symptom in IBD. The pathogenesis of IBD-associated diarrhea is multifactorial and is essentially an outcome of mucosal damage caused by persistent inflammation resulting in dysregulated intestinal ion transport, impaired epithelial barrier function and increased accessibility of the pathogens to the intestinal mucosa. Altered expression and/or function of epithelial ion transporters and channels is the principle cause of electrolyte retention and water accumulation in the intestinal lumen leading to diarrhea in IBD. Aberrant barrier function further contributes to diarrhea via leak-flux mechanism. Mucosal penetration of enteric pathogens promotes dysbiosis and exacerbates the underlying immune system further perpetuating IBD associated-tissue damage and diarrhea. Here, we review the mechanisms of impaired ion transport and loss of epithelial barrier function contributing to diarrhea associated with IBD.
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Affiliation(s)
- Arivarasu N Anbazhagan
- a Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago , Chicago , IL , USA
| | - Shubha Priyamvada
- a Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago , Chicago , IL , USA
| | - Waddah A Alrefai
- a Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago , Chicago , IL , USA.,b Jesse Brown VA Medical Center , Chicago , IL , USA
| | - Pradeep K Dudeja
- a Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago , Chicago , IL , USA.,b Jesse Brown VA Medical Center , Chicago , IL , USA
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21
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Schwartz L, Lafitte O, da Veiga Moreira J. Toward a Reasoned Classification of Diseases Using Physico-Chemical Based Phenotypes. Front Physiol 2018. [PMID: 29541031 PMCID: PMC5835834 DOI: 10.3389/fphys.2018.00094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Background: Diseases and health conditions have been classified according to anatomical site, etiological, and clinical criteria. Physico-chemical mechanisms underlying the biology of diseases, such as the flow of energy through cells and tissues, have been often overlooked in classification systems. Objective: We propose a conceptual framework toward the development of an energy-oriented classification of diseases, based on the principles of physical chemistry. Methods: A review of literature on the physical chemistry of biological interactions in a number of diseases is traced from the point of view of the fluid and solid mechanics, electricity, and chemistry. Results: We found consistent evidence in literature of decreased and/or increased physical and chemical forces intertwined with biological processes of numerous diseases, which allowed the identification of mechanical, electric and chemical phenotypes of diseases. Discussion: Biological mechanisms of diseases need to be evaluated and integrated into more comprehensive theories that should account with principles of physics and chemistry. A hypothetical model is proposed relating the natural history of diseases to mechanical stress, electric field, and chemical equilibria (ATP) changes. The present perspective toward an innovative disease classification may improve drug-repurposing strategies in the future.
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Affiliation(s)
| | - Olivier Lafitte
- LAGA, UMR 7539, Paris 13 University, Sorbonne Paris Cité, Villetaneuse, France
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22
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Peña-Oyarzun D, Troncoso R, Kretschmar C, Hernando C, Budini M, Morselli E, Lavandero S, Criollo A. Hyperosmotic stress stimulates autophagy via polycystin-2. Oncotarget 2017; 8:55984-55997. [PMID: 28915568 PMCID: PMC5593539 DOI: 10.18632/oncotarget.18995] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2017] [Accepted: 06/21/2017] [Indexed: 12/13/2022] Open
Abstract
Various intracellular mechanisms are activated in response to stress, leading to adaptation or death. Autophagy, an intracellular process that promotes lysosomal degradation of proteins, is an adaptive response to several types of stress. Osmotic stress occurs under both physiological and pathological conditions, provoking mechanical stress and activating various osmoadaptive mechanisms. Polycystin-2 (PC2), a membrane protein of the polycystin family, is a mechanical sensor capable of activating the cell signaling pathways required for cell adaptation and survival. Here we show that hyperosmotic stress provoked by treatment with hyperosmolar concentrations of sorbitol or mannitol induces autophagy in HeLa and HCT116 cell lines. In addition, we show that mTOR and AMPK, two stress sensor proteins involved modulating autophagy, are downregulated and upregulated, respectively, when cells are subjected to hyperosmotic stress. Finally, our findings show that PC2 is required to promote hyperosmotic stress-induced autophagy. Downregulation of PC2 prevents inhibition of hyperosmotic stress-induced mTOR pathway activation. In conclusion, our data provide new insight into the role of PC2 as a mechanosensor that modulates autophagy under hyperosmotic stress conditions.
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Affiliation(s)
- Daniel Peña-Oyarzun
- Advanced Center for Chronic Diseases, Facultad Ciencias Quimicas y Farmaceuticas & Facultad Medicina, Universidad de Chile, Santiago, Chile.,Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile
| | - Rodrigo Troncoso
- Advanced Center for Chronic Diseases, Facultad Ciencias Quimicas y Farmaceuticas & Facultad Medicina, Universidad de Chile, Santiago, Chile.,Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Chile
| | - Catalina Kretschmar
- Advanced Center for Chronic Diseases, Facultad Ciencias Quimicas y Farmaceuticas & Facultad Medicina, Universidad de Chile, Santiago, Chile.,Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Santiago, Chile
| | - Cecilia Hernando
- Advanced Center for Chronic Diseases, Facultad Ciencias Quimicas y Farmaceuticas & Facultad Medicina, Universidad de Chile, Santiago, Chile.,Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Santiago, Chile
| | - Mauricio Budini
- Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Santiago, Chile
| | - Eugenia Morselli
- Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Sergio Lavandero
- Advanced Center for Chronic Diseases, Facultad Ciencias Quimicas y Farmaceuticas & Facultad Medicina, Universidad de Chile, Santiago, Chile.,Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile.,Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Alfredo Criollo
- Advanced Center for Chronic Diseases, Facultad Ciencias Quimicas y Farmaceuticas & Facultad Medicina, Universidad de Chile, Santiago, Chile.,Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Santiago, Chile
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23
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Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress. Mol Cell Biol 2017; 37:MCB.00521-16. [PMID: 27920257 DOI: 10.1128/mcb.00521-16] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2016] [Accepted: 12/01/2016] [Indexed: 12/25/2022] Open
Abstract
High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1β. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.
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24
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Changes in the Luminal Environment of the Colonic Epithelial Cells and Physiopathological Consequences. THE AMERICAN JOURNAL OF PATHOLOGY 2017; 187:476-486. [PMID: 28082121 DOI: 10.1016/j.ajpath.2016.11.015] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Revised: 11/21/2016] [Accepted: 11/23/2016] [Indexed: 12/28/2022]
Abstract
Evidence, mostly from experimental models, has accumulated, indicating that modifications of bacterial metabolite concentrations in the large intestine luminal content, notably after changes in the dietary composition, may have important beneficial or deleterious consequences for the colonic epithelial cell metabolism and physiology in terms of mitochondrial energy metabolism, reactive oxygen species production, gene expression, DNA integrity, proliferation, and viability. Recent data suggest that for some bacterial metabolites, like hydrogen sulfide and butyrate, the extent of their oxidation in colonocytes affects their capacity to modulate gene expression in these cells. Modifications of the luminal bacterial metabolite concentrations may, in addition, affect the colonic pH and osmolarity, which are known to affect colonocyte biology per se. Although the colonic epithelium appears able to face, up to some extent, changes in its luminal environment, notably by developing a metabolic adaptive response, some of these modifications may likely affect the homeostatic process of colonic epithelium renewal and the epithelial barrier function. The contribution of major changes in the colonocyte luminal environment in pathological processes, like mucosal inflammation, preneoplasia, and neoplasia, although suggested by several studies, remains to be precisely evaluated, particularly in a long-term perspective.
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Amara S, Alotaibi D, Tiriveedhi V. NFAT5/STAT3 interaction mediates synergism of high salt with IL-17 towards induction of VEGF-A expression in breast cancer cells. Oncol Lett 2016; 12:933-943. [PMID: 27446373 PMCID: PMC4950837 DOI: 10.3892/ol.2016.4713] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2015] [Accepted: 05/23/2016] [Indexed: 12/29/2022] Open
Abstract
Chronic inflammation has been considered an important player in cancer proliferation and progression. High salt (sodium chloride) levels have been considered a potent inducer of chronic inflammation. In the present study, the synergistic role of high salt with interleukin (IL)-17 towards induction of the inflammatory and angiogenic stress factor vascular endothelial growth factor (VEGF)-A was investigated. Stimulation of MCF-7 breast cancer cells with high salt (0.2 M NaCl) and sub-minimal IL-17 (1 ng/ml) enhanced the expression of VEGF-A (2.9 and 2.6-fold, respectively, P<0.05) compared with untreated cells. Furthermore, co-treatment with both high salt and sub-minimal IL-17 led to a 5.9-fold increase in VEGF-A expression (P<0.01), thus suggesting a synergistic role of these factors. VEGF-A promoter analysis and specific small interfering RNA knock-down of transcription factors revealed that high salt induced VEGF-A expression through nuclear factor of activated T-cells (NFAT)5, while IL-17 induced VEGF-A expression via signal transducer and activator of transcription (STAT)3 signaling mechanisms. Treatment of normal human aortic endothelial cells with the supernatant of activated MCF-7 cells enhanced cell migration and induced expression of migration-specific factors, including vascular cell adhesion protein, β1 integrin and cluster of differentiation 31. These data suggest that high salt levels synergize with pro-inflammatory IL-17 to potentially induce cancer progression and metastasis through VEGF-A expression. Therefore, low-salt diet, anti-NFAT5 and anti-STAT3 therapies may provide novel avenues for enhanced efficiency of the current cancer therapy.
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Affiliation(s)
- Suneetha Amara
- Department of Medicine, Mercy Hospital, St. Louis, MO 63141, USA
| | - Dalal Alotaibi
- Department of Biological Sciences, Tennessee State University, Nashville, TN 37209, USA
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Amara S, Ivy MT, Myles EL, Tiriveedhi V. Sodium channel γENaC mediates IL-17 synergized high salt induced inflammatory stress in breast cancer cells. Cell Immunol 2015; 302:1-10. [PMID: 26723502 DOI: 10.1016/j.cellimm.2015.12.007] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2015] [Revised: 12/15/2015] [Accepted: 12/18/2015] [Indexed: 01/03/2023]
Abstract
Chronic inflammation is known to play a critical role in the development of cancer. Recent evidence suggests that high salt in the tissue microenvironment induces chronic inflammatory milieu. In this report, using three breast cancer-related cell lines, we determined the molecular basis of the potential synergistic inflammatory effect of sodium chloride (NaCl) with interleukin-17 (IL-17). Combined treatment of high NaCl (0.15M) with sub-effective IL-17 (0.1 nM) induced enhanced growth in breast cancer cells along with activation of reactive nitrogen and oxygen (RNS/ROS) species known to promote cancer. Similar effect was not observed with equi-molar mannitol. This enhanced of ROS/RNS activity correlates with upregulation of γENaC an inflammatory sodium channel. The similar culture conditions have also induced expression of pro-inflammatory cytokines such as IL-6, TNFα etc. Taken together, these data suggest that high NaCl in the cellular microenvironment induces a γENaC mediated chronic inflammatory response with a potential pro-carcinogenic effect.
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Affiliation(s)
- Suneetha Amara
- Department of Medicine, Mercy Hospital, St Louis, MO, USA
| | - Michael T Ivy
- Department of Biological Sciences, Tennessee State University, Nashville, TN, USA
| | - Elbert L Myles
- Department of Biological Sciences, Tennessee State University, Nashville, TN, USA
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Priyamvada S, Gomes R, Gill RK, Saksena S, Alrefai WA, Dudeja PK. Mechanisms Underlying Dysregulation of Electrolyte Absorption in Inflammatory Bowel Disease-Associated Diarrhea. Inflamm Bowel Dis 2015; 21:2926-35. [PMID: 26595422 PMCID: PMC4662046 DOI: 10.1097/mib.0000000000000504] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Inflammatory bowel diseases (IBDs), including Crohn's disease and ulcerative colitis, are chronic relapsing inflammatory disorders of the gastrointestinal tract. Chronic inflammation of the intestine affects the normal fluid and electrolyte absorption leading to diarrhea, the hallmark symptom of IBD. The management of IBD-associated diarrhea still remains to be a challenge, and extensive studies over the last 2 decades have focused on investigating the molecular mechanisms underlying IBD-associated diarrhea. These studies have shown that the predominant mechanism of diarrhea in IBD involves impairment of electroneutral NaCl absorption, with very little role if any played by anion secretion. The electroneutral NaCl absorption involves coupled operation of Na/H exchanger 3 (NHE3 or SLC9A3) and Cl/HCO3 exchanger DRA (Down Regulated in Adenoma, or SLC26A3). Increasing evidence now supports the critical role of a marked decrease in NHE3 and DRA function and/or expression in IBD-associated diarrhea. This review provides a detailed analysis of the current knowledge related to alterations in NHE3 and DRA function and expression in IBD including the mechanisms underlying these observations and highlights the potential of these transporters as important and novel therapeutic targets.
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Affiliation(s)
- Shubha Priyamvada
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL
| | - Rochelle Gomes
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL
| | - Ravinder K. Gill
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL
| | - Seema Saksena
- Jesse Brown VA Medical Center, University of Illinois at Chicago, Chicago, IL
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL
| | - Waddah A. Alrefai
- Jesse Brown VA Medical Center, University of Illinois at Chicago, Chicago, IL
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL
| | - Pradeep K. Dudeja
- Jesse Brown VA Medical Center, University of Illinois at Chicago, Chicago, IL
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL
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Boland BS, Widjaja CE, Banno A, Zhang B, Kim SH, Stoven S, Peterson MR, Jones MC, Su HI, Crowe SE, Bui JD, Ho SB, Okugawa Y, Goel A, Marietta EV, Khosroheidari M, Jepsen K, Aramburu J, López-Rodríguez C, Sandborn WJ, Murray JA, Harismendy O, Chang JT. Immunodeficiency and autoimmune enterocolopathy linked to NFAT5 haploinsufficiency. THE JOURNAL OF IMMUNOLOGY 2015; 194:2551-60. [PMID: 25667416 DOI: 10.4049/jimmunol.1401463] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The link between autoimmune diseases and primary immunodeficiency syndromes has been increasingly appreciated. Immunologic evaluation of a young man with autoimmune enterocolopathy and unexplained infections revealed evidence of immunodeficiency, including IgG subclass deficiency, impaired Ag-induced lymphocyte proliferation, reduced cytokine production by CD8(+) T lymphocytes, and decreased numbers of NK cells. Genetic evaluation identified haploinsufficiency of NFAT5, a transcription factor regulating immune cell function and cellular adaptation to hyperosmotic stress, as a possible cause of this syndrome. Inhibition or deletion of NFAT5 in normal human and murine cells recapitulated several of the immune deficits identified in the patient. These results provide evidence of a primary immunodeficiency disorder associated with organ-specific autoimmunity linked to NFAT5 deficiency.
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Affiliation(s)
- Brigid S Boland
- Department of Medicine, University of California San Diego, La Jolla, CA 92093; Inflammatory Bowel Disease Center, University of California San Diego, La Jolla, CA 92093
| | | | - Asoka Banno
- Department of Medicine, University of California San Diego, La Jolla, CA 92093
| | - Bing Zhang
- Department of Medicine, University of California San Diego, La Jolla, CA 92093
| | - Stephanie H Kim
- Department of Medicine, University of California San Diego, La Jolla, CA 92093
| | | | - Michael R Peterson
- Western Washington Pathology and Multicare Health System, Tacoma, WA 98405
| | - Marilyn C Jones
- Department of Pediatrics and Rady Children's Hospital, University of California San Diego, La Jolla, CA 92093
| | - H Irene Su
- Department of Reproductive Medicine, University of California San Diego, La Jolla, CA 92093
| | - Sheila E Crowe
- Department of Medicine, University of California San Diego, La Jolla, CA 92093
| | - Jack D Bui
- Department of Pathology, University of California San Diego, La Jolla, CA 92093
| | - Samuel B Ho
- Department of Pathology, University of California San Diego, La Jolla, CA 92093
| | - Yoshinaga Okugawa
- Center for Gastrointestinal Research, Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Research Institute, Baylor University Medical Center, Dallas, TX 75246; Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX 75246
| | - Ajay Goel
- Center for Gastrointestinal Research, Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Research Institute, Baylor University Medical Center, Dallas, TX 75246; Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX 75246
| | | | - Mahdieh Khosroheidari
- Institute of Genomic Medicine, University of California San Diego, La Jolla, CA 92093
| | - Kristen Jepsen
- Institute of Genomic Medicine, University of California San Diego, La Jolla, CA 92093
| | - Jose Aramburu
- Immunology Unit, Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona 08003 Spain; and
| | - Cristina López-Rodríguez
- Immunology Unit, Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona 08003 Spain; and
| | - William J Sandborn
- Department of Medicine, University of California San Diego, La Jolla, CA 92093; Inflammatory Bowel Disease Center, University of California San Diego, La Jolla, CA 92093
| | | | - Olivier Harismendy
- Department of Medicine, University of California San Diego, La Jolla, CA 92093; Moores Cancer Center, University of California San Diego, La Jolla, CA 92093
| | - John T Chang
- Department of Medicine, University of California San Diego, La Jolla, CA 92093; Inflammatory Bowel Disease Center, University of California San Diego, La Jolla, CA 92093;
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Juste C, Kreil DP, Beauvallet C, Guillot A, Vaca S, Carapito C, Mondot S, Sykacek P, Sokol H, Blon F, Lepercq P, Levenez F, Valot B, Carré W, Loux V, Pons N, David O, Schaeffer B, Lepage P, Martin P, Monnet V, Seksik P, Beaugerie L, Ehrlich SD, Gibrat JF, Van Dorsselaer A, Doré J. Bacterial protein signals are associated with Crohn's disease. Gut 2014; 63:1566-77. [PMID: 24436141 PMCID: PMC4173658 DOI: 10.1136/gutjnl-2012-303786] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
OBJECTIVE No Crohn's disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. DESIGN We first developed and validated a workflow-including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS-to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. RESULTS Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. CONCLUSIONS This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis.
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Affiliation(s)
| | - David P Kreil
- Chair of Bioinformatics, Boku University Vienna, Vienna, Austria,Department of Life Sciences, University of Warwick, Warwickshire, UK
| | | | - Alain Guillot
- Plate-forme d'Analyse Protéomique de Paris Sud-Ouest (PAPPSO), INRA, Gif-sur-Yvette, France
| | - Sebastian Vaca
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France
| | - Christine Carapito
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France
| | | | - Peter Sykacek
- Chair of Bioinformatics, Boku University Vienna, Vienna, Austria
| | - Harry Sokol
- UMR1319 Micalis, INRA, Jouy-en-Josas, France,Gastroenterology and Nutrition Unit, Hôpital Saint-Antoine, AP-HP, Paris, France
| | | | | | | | - Benoît Valot
- Plate-forme d'Analyse Protéomique de Paris Sud-Ouest (PAPPSO), INRA, Gif-sur-Yvette, France
| | - Wilfrid Carré
- UR1077, Mathématique Informatique et Génome (MIG), INRA, Jouy-en-Josas, France
| | - Valentin Loux
- UR1077, Mathématique Informatique et Génome (MIG), INRA, Jouy-en-Josas, France
| | | | - Olivier David
- UR341, Mathématiques et Informatique Appliquées (MIA), INRA, Jouy-en-Josas, France
| | - Brigitte Schaeffer
- UR341, Mathématiques et Informatique Appliquées (MIA), INRA, Jouy-en-Josas, France
| | | | - Patrice Martin
- UMR1313 GABI, Iso Cell Express (ICE), INRA, Jouy-en-Josas, France
| | | | - Philippe Seksik
- Gastroenterology and Nutrition Unit, Hôpital Saint-Antoine, AP-HP, Paris, France
| | - Laurent Beaugerie
- Gastroenterology and Nutrition Unit, Hôpital Saint-Antoine, AP-HP, Paris, France
| | | | | | - Alain Van Dorsselaer
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France
| | - Joël Doré
- UMR1319 Micalis, INRA, Jouy-en-Josas, France
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Zhou X, Wang H, Burg MB, Ferraris JD. Inhibitory phosphorylation of GSK-3β by AKT, PKA, and PI3K contributes to high NaCl-induced activation of the transcription factor NFAT5 (TonEBP/OREBP). Am J Physiol Renal Physiol 2013; 304:F908-17. [PMID: 23324178 DOI: 10.1152/ajprenal.00591.2012] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
High NaCl activates the transcription factor nuclear factor of activated T cells 5 (NFAT5), leading to increased transcription of osmoprotective target genes. Kinases PKA, PI3K, AKT1, and p38α were known to contribute to the high NaCl-induced increase of NFAT5 activity. We now identify another kinase, GSK-3β. siRNA-mediated knock-down of GSK-3β increases NFAT5 transcriptional and transactivating activities without affecting high NaCl-induced nuclear localization of NFAT5 or NFAT5 protein expression. High NaCl increases phosphorylation of GSK-3β-S9, which inhibits GSK-3β. In GSK-3β-null mouse embryonic fibroblasts transfection of GSK-3β, in which serine 9 is mutated to alanine, so that it cannot be inhibited by phosphorylation at that site, inhibits high NaCl-induced NFAT5 transcriptional activity more than transfection of wild-type GSK-3β. High NaCl-induced phosphorylation of GSK-3β-S9 depends on PKA, PI3K, and AKT, but not p38α. Overexpression of PKA catalytic subunit α or of catalytically active AKT1 reduces inhibition of NFAT5 by GSK-3β, but overexpression of p38α together with its catalytically active upstream kinase, MKK6, does not. Thus, GSK-3β normally inhibits NFAT5 by suppressing its transactivating activity. When activated by high NaCl, PKA, PI3K, and AKT1, but not p38α, increase phosphorylation of GSK-3β-S9, which reduces the inhibitory effect of GSK-3β on NFAT5, and thus contributes to activation of NFAT5.
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Affiliation(s)
- Xiaoming Zhou
- Department of Medicine, Uniformed Services University, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA.
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31
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Barkas F, Liberopoulos E, Kei A, Elisaf M. Electrolyte and acid-base disorders in inflammatory bowel disease. Ann Gastroenterol 2013; 26:23-28. [PMID: 24714322 PMCID: PMC3959504] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2012] [Accepted: 04/02/2012] [Indexed: 11/03/2022] Open
Abstract
INFLAMMATORY BOWEL DISEASE (IBD) IS A CHRONIC INFLAMMATORY INTESTINAL DISORDER ENCOMPASSING TWO MAJOR ENTITIES: Crohn's disease and ulcerative colitis. Intestinal inflammatory processes reduce the absorption of sodium, chloride and calcium, while they increase potassium secretion. In addition, mild to severe metabolic alkalosis may occur in IBD patients, mainly depending on the severity of the disease and the part of the gastrointestinal tract being affected. The aim of this review is the presentation of the electrolyte and acid-base disturbances in IBD and how the activity state of the disease and/or treatment may affect them.
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Affiliation(s)
- Fotis Barkas
- Department of Internal Medicine, University of Ioannina Medical School, Ioannina, Greece
| | - Evangelos Liberopoulos
- Department of Internal Medicine, University of Ioannina Medical School, Ioannina, Greece,
Correspondence to: Evangelos Liberopoulos, MD FASA FRSH, Ass. Professor of Internal Medicine, University of Ioannina Medical School, 45 110 Ioannina, Greece, Tel.: +30 26510 07502, Fax: +30 26510 07016, e-mail:
| | - Anastazia Kei
- Department of Internal Medicine, University of Ioannina Medical School, Ioannina, Greece
| | - Moses Elisaf
- Department of Internal Medicine, University of Ioannina Medical School, Ioannina, Greece
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Brocker C, Thompson DC, Vasiliou V. The role of hyperosmotic stress in inflammation and disease. Biomol Concepts 2012; 3:345-364. [PMID: 22977648 PMCID: PMC3438915 DOI: 10.1515/bmc-2012-0001] [Citation(s) in RCA: 202] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hyperosmotic stress is an often overlooked process that potentially contributes to a number of human diseases. Whereas renal hyperosmolarity is a well-studied phenomenon, recent research provides evidence that many non-renal tissues routinely experience hyperosmotic stress that may contribute significantly to disease initiation and progression. Moreover, a growing body of evidence implicates hyperosmotic stress as a potent inflammatory stimulus by triggering proinflammatory cytokine release and inflammation. Under physiological conditions, the urine concentrating mechanism within the inner medullary region of the mammalian kidney exposes cells to high extracellular osmolarity. As such, renal cells have developed many adaptive strategies to compensate for increased osmolarity. Hyperosmotic stress is linked to many maladies, including acute and chronic, as well as local and systemic, inflammatory disorders. Hyperosmolarity triggers cell shrinkage, oxidative stress, protein carbonylation, mitochondrial depolarization, DNA damage, and cell cycle arrest, thus rendering cells susceptible to apoptosis. However, many adaptive mechanisms exist to counter the deleterious effects of hyperosmotic stress, including cytoskeletal rearrangement and up-regulation of antioxidant enzymes, transporters, and heat shock proteins. Osmolyte synthesis is also up-regulated and many of these compounds have been shown to reduce inflammation. The cytoprotective mechanisms and associated regulatory pathways that accompany the renal response to hyperosmolarity are found in many non-renal tissues, suggesting cells are commonly confronted with hyperosmotic conditions. Osmoadaptation allows cells to survive and function under potentially cytotoxic conditions. This review covers the pathological consequences of hyperosmotic stress in relation to disease and emphasizes the importance of considering hyperosmolarity in inflammation and disease progression.
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Affiliation(s)
- Chad Brocker
- Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - David C. Thompson
- Department of Clinical Pharmacy, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Vasilis Vasiliou
- Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
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Neuhofer W. Role of NFAT5 in inflammatory disorders associated with osmotic stress. Curr Genomics 2011; 11:584-90. [PMID: 21629436 PMCID: PMC3078683 DOI: 10.2174/138920210793360961] [Citation(s) in RCA: 98] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2010] [Revised: 10/05/2010] [Accepted: 10/11/2010] [Indexed: 12/25/2022] Open
Abstract
Nuclear factor of activated T cells 5 (NFAT5) is the most recently described member of the Rel family of transcription factors, including NF-κB and NFAT1-4, which play central roles in inducible gene expression during the immune response. NFAT5 was initially described to drive osmoprotective gene expression in renal medullary cells, which are routinely faced by high extracellular osmolalities. Recent data however indicate profound biological importance of the mammalian osmotic stress response in view of NFAT5 dependent gene regulation in non-renal tissues. In mononuclear cells and epithelial cells, NFAT5 stimulates the expression of various pro-inflammatory cytokines during elevated ambient tonicity. Accordingly, compared to plasma, the interstitial tonicity of lymphoid organs like spleen and thymus and that of liver is substantially hypertonic under physiological conditions. In addition, anisotonic disorders (hypernatremia, diabetes mellitus, dehydration) entail systemic hyperosmolality, and, in inflammatory disorders, the skin, intestine, and cornea are sites of local hyperosmolality. This article summarizes the current knowledge regarding systemic and local osmotic stress in anisotonic and inflammatory disorders in view of NFAT5 activation and regulation, and NFAT5 dependent cytokine production.
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Affiliation(s)
- Wolfgang Neuhofer
- Departments of Nephrology and Physiology, Inner City Campus, University of Munich, Munich, Germany
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Samak G, Narayanan D, Jaggar JH, Rao R. CaV1.3 channels and intracellular calcium mediate osmotic stress-induced N-terminal c-Jun kinase activation and disruption of tight junctions in Caco-2 CELL MONOLAYERS. J Biol Chem 2011; 286:30232-43. [PMID: 21737448 DOI: 10.1074/jbc.m111.240358] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
We investigated the role of a Ca(2+) channel and intracellular calcium concentration ([Ca(2+)](i)) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-mediated intracellular Ca(2+) depletion. Depletion of extracellular Ca(2+) at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca(2+) depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca(2+) depletion partially reduced osmotic stress-induced rise in [Ca(2+)](i), whereas thapsigargin and extracellular Ca(2+) depletion together resulted in almost complete loss of rise in [Ca(2+)](i). L-type Ca(2+) channel blockers (isradipine and diltiazem) or knockdown of the Ca(V)1.3 channel abrogated [Ca(2+)](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca(2+) depletion, thapsigargin, or Ca(V)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca(2+) depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca(V)1.3 and rise in [Ca(2+)](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca(2+)](i) mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca(2+) also contributes to osmotic stress-induced tight junction disruption.
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Affiliation(s)
- Geetha Samak
- Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA
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35
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Farkas K, Yeruva S, Rakonczay Z, Ludolph L, Molnár T, Nagy F, Szepes Z, Schnúr A, Wittmann T, Hubricht J, Riederer B, Venglovecz V, Lázár G, Király M, Zsembery Á, Varga G, Seidler U, Hegyi P. New therapeutic targets in ulcerative colitis: the importance of ion transporters in the human colon. Inflamm Bowel Dis 2011; 17:884-898. [PMID: 20722063 DOI: 10.1002/ibd.21432] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2010] [Accepted: 06/22/2010] [Indexed: 12/12/2022]
Abstract
BACKGROUND The absorption of water and ions (especially Na(+) and Cl(-)) is an important function of colonic epithelial cells in both physiological and pathophysiological conditions. Despite the comprehensive animal studies, there are only scarce available data on the ion transporter activities of the normal and inflamed human colon. METHODS In this study, 128 healthy controls and 69 patients suffering from ulcerative colitis (UC) were involved. We investigated the expressional and functional characteristics of the Na(+)/H(+) exchangers (NHE) 1-3, the epithelial sodium channel (ENaC), and the SLC26A3 Cl(-)/HCO 3- exchanger downregulated in adenoma (DRA) in primary colonic crypts isolated from human biopsy and surgical samples using microfluorometry, patch clamp, and real-time reverse-transcription polymerase chain reaction (RT-PCR) techniques. RESULTS Data collected from colonic crypts showed that the activities of electroneutral (via NHE3) and the electrogenic Na(+) absorption (via ENaC) are in inverse ratio to each other in the proximal and distal colon. We found no significant differences in the activity of NHE2 in different segments of the colon. Surface cell Cl(-)/HCO 3- exchange is more active in the distal part of the colon. Importantly, both sodium and chloride absorptions are damaged in UC, whereas NHE1, which has been shown to promote immune response, is upregulated by 6-fold. CONCLUSIONS These results open up new therapeutic targets in UC.
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Affiliation(s)
- Klaudia Farkas
- First Department of Medicine, University of Szeged, Szeged, Hungary
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Samak G, Suzuki T, Bhargava A, Rao RK. c-Jun NH2-terminal kinase-2 mediates osmotic stress-induced tight junction disruption in the intestinal epithelium. Am J Physiol Gastrointest Liver Physiol 2010; 299:G572-84. [PMID: 20595622 PMCID: PMC3774214 DOI: 10.1152/ajpgi.00265.2010] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Gastrointestinal epithelium faces osmotic stress, both at physiological and pathophysiological conditions. JNK activation is an immediate cellular response to osmotic stress. We investigated the effect of osmotic stress on intestinal epithelial barrier function and delineated the role of JNK2 in osmotic stress-induced tight junction (TJ) regulation in Caco-2 cell monolayers and ileum of Jnk(-/-) and Jnk2(-/-) mice. The role of JNK activation in osmotic stress-induced TJ disruption was evaluated using JNK-specific inhibitor and antisense oligonucleotides. Furthermore, the effect of cold restraint stress in vivo on TJ integrity was determined in rats. Osmotic stress disrupted TJs and barrier function in Caco-2 cell monolayers without affecting cell viability. Osmotic stress activated JNK1 and JNK2 and the inhibition of JNK by SP600125 attenuated osmotic stress-induced TJ disruption. TJ disruption and barrier dysfunction by osmotic stress was associated with JNK-dependent remodeling of actin cytoskeleton. Knockdown of JNK2 accelerated TJ assembly and attenuated osmotic stress-induced TJ disruption in Caco-2 cell monolayers. In mouse ileum in vitro, osmotic stress increased paracellular permeability, which was attenuated by SP600125. Osmotic stress disrupted actin cytoskeleton and TJs and increased paracellular permeability in the ileum of wild-type and JNK1(-/-) mice, but not in JNK2(-/-) mouse ileum. Cold restraint stress activated JNK in rat ileum and caused JNK-dependent remodeling of actin cytoskeleton and redistribution of occludin and zona occluden-1 from the intercellular junctions. These results reveal the role of JNK2 in the mechanism of osmotic stress-induced TJ disruption in the intestinal epithelium.
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Affiliation(s)
- G. Samak
- 1Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee; ,2Department of Zoology, DVS College, Shimoga, India; and
| | - T. Suzuki
- 1Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee;
| | - A. Bhargava
- 3Department of Surgery, University of California, San Francisco, California
| | - R. K. Rao
- 1Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee;
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Oxley APA, Lanfranconi MP, Würdemann D, Ott S, Schreiber S, McGenity TJ, Timmis KN, Nogales B. Halophilic archaea in the human intestinal mucosa. Environ Microbiol 2010; 12:2398-410. [PMID: 20438582 DOI: 10.1111/j.1462-2920.2010.02212.x] [Citation(s) in RCA: 89] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The human gastrointestinal tract microbiota, despite its key roles in health and disease, remains a diverse, variable and poorly understood entity. Current surveys reveal a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea. In an analysis of the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease we found 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae (haloarchaea), including novel phylotypes. As the human colon is not considered a salty environment and haloarchaea are described as extreme halophiles, we evaluated and further discarded the possibility that these sequences originated from pre-colonoscopy saline lavage solutions. Furthermore, aerobic enrichment cultures prepared from a patient biopsy at low salinity (2.5% NaCl) yielded haloarchaeal sequence types. Microscopic observation after fluorescence in situ hybridization provided evidence of the presence of viable archaeal cells in these cultures. These results prove the survival of haloarchaea in the digestive system and suggest that they may be members of the mucosal microbiota, even if present in low numbers in comparison with methanogenic archaea. Investigation of a potential physiological basis of this association may lead to new insights into gastrointestinal health and disease.
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Affiliation(s)
- Andrew P A Oxley
- Environmental Microbiology Laboratory, Helmholtz Centre for Infection Research, Braunschweig, Germany.
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Schwartz L, Guais A, Pooya M, Abolhassani M. Is inflammation a consequence of extracellular hyperosmolarity? JOURNAL OF INFLAMMATION-LONDON 2009; 6:21. [PMID: 19549308 PMCID: PMC2709204 DOI: 10.1186/1476-9255-6-21] [Citation(s) in RCA: 78] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/13/2009] [Accepted: 06/23/2009] [Indexed: 11/10/2022]
Abstract
Background There are several reports suggesting that hyperosmolarity induces inflammation. We recently showed that Dextran Sodium Sulfate causes inflammatory bowel disease due to hyperosmolarity. The aim of this study was to confirm the link between hyperosmolarity and inflammation by assessing osmolarity values in vivo during inflammation, compare the inflammatory potential of different osmotic agents and finally study the long-term consequences of hyperosmolarity on cell fate. Methods Osmotic pressures were measured in inflammatory liquids withdrawn from mice subjected to inflammation caused either by subcutaneous injection of Bacille Calmette-Guérin (BCG) or Freund adjuvant. Three epithelial cell lines (HT29, T24 and A549) were exposed up to 48 hours to increasing osmolarities (300, 600, 900 mOsm) of chemically inert molecules such as Mannitol, Propylene Glycol, and Glycerol and inflammatory response was assessed by Enzyme Linked ImmunoSorbent Assay (ELISA) and RNA Protection Assay (RPA). Finally, normal mouse macrophages were exposed to hyperosmotic conditions for long-term culture. Results The inflammation caused either by BCG or Freund adjuvant is correlated to hyperosmolarity in inflammatory liquids. The exposure of cells to the different compounds, whatever their molecular weight, has no effect on the secretion of cytokines as long as the osmolarity is below a threshold of 300 mOsm. Higher osmolarities result in the secretion of proinflammatory cytokines (Interleukin-8, Interleukin-6, Interleukin-1β and Tumor Necrosis factor-α). Long-term hyperosmotic culture extends normal macrophage half-life, from 44 days to 102 days, and alters the expression of p53, Bcl-2 and Bax. Conclusion The present study further suggests inflammation and hyperosmolarity are closely related phenomena if not synonymous.
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Affiliation(s)
- Laurent Schwartz
- Service de Radiothérapie Hôpital Pitié-Salpétrière, Paris, 75013 France.
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Ste20-related proline/alanine-rich kinase (SPAK) regulated transcriptionally by hyperosmolarity is involved in intestinal barrier function. PLoS One 2009; 4:e5049. [PMID: 19343169 PMCID: PMC2660421 DOI: 10.1371/journal.pone.0005049] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2009] [Accepted: 02/06/2009] [Indexed: 01/01/2023] Open
Abstract
The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-kappaB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-kappaB and Sp1 to bind to their binding sites. Knock-down of either NF-kappaB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-kappaB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function.
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Schwartz L, Abolhassani M, Pooya M, Steyaert JM, Wertz X, Israël M, Guais A, Chaumet-Riffaud P. Hyperosmotic stress contributes to mouse colonic inflammation through the methylation of protein phosphatase 2A. Am J Physiol Gastrointest Liver Physiol 2008; 295:G934-41. [PMID: 18755808 DOI: 10.1152/ajpgi.90296.2008] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
There are several reports suggesting hyperosmotic contents in the feces of patients suffering from inflammatory bowel disease (IBD). Previous works have documented that hyperosmolarity can cause inflammation attributable to methylation of the catalytic subunit of protein phosphatase 2A (PP2A) and subsequent NF-kappaB activation resulting in cytokine secretion. In this study, we demonstrate that dextran sulfate sodium (DSS) induces colitis due to hyperosmolarity and subsequent PP2A activation. Mice were randomized and fed with increased concentrations of DSS (0 mOsm, 175 mOsm, 300 mOsm, and 627 mOsm) for a duration of 3 wk or with hyperosmotic concentrations of DSS (627 mOsm) or mannitol (450 mOsm) for a duration of 12 wk. Long-term oral administration of hyposmotic DSS or mannitol had no demonstrable effect. Hyperosmotic DSS or mannitol produced a significant increase in colonic inflammation, as well as an increase in the weight of sacral lymph nodes and in serum amyloid A protein levels. Similar results were obtained through the ingestion of comparable osmolarities of mannitol. Hyperosmolarity induces the methylation of PP2A, nuclear p65 NF-kappaB activation. and cytokine secretion. The rectal instillation of okadaic acid, a well-known PP2A inhibitor, reverses the IBD. Short inhibiting RNAs (siRNAs) targeted toward PP2Ac reverse the effect of hyperosmotic DSS. The present study strongly suggests that DSS-induced chronic colitis is a consequence of the methylation of PP2Ac induced by hyperosmolarity.
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Affiliation(s)
- Laurent Schwartz
- Service de Radiothérapie Hôpital Pitié-Salpétrière, bd. de l'Hôpital, 75013 Paris, France.
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Lunn JA, Jacamo R, Rozengurt E. Preferential Phosphorylation of Focal Adhesion Kinase Tyrosine 861 Is Critical for Mediating an Anti-apoptotic Response to Hyperosmotic Stress. J Biol Chem 2007; 282:10370-9. [PMID: 17289681 DOI: 10.1074/jbc.m607780200] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress.
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Affiliation(s)
- J Adrian Lunn
- Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, UCLA-CURE, Digestive Diseases Research Center and Molecular Biology Institute, UCLA, Los Angeles, California 90095
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42
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De Vito P. The sodium/hydrogen exchanger: a possible mediator of immunity. Cell Immunol 2006; 240:69-85. [PMID: 16930575 DOI: 10.1016/j.cellimm.2006.07.001] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2006] [Revised: 06/30/2006] [Accepted: 07/05/2006] [Indexed: 10/24/2022]
Abstract
Immune cells such as macrophages and neutrophils provide the first line of defence of the immune system using phagocytosis, cytokine and chemokine synthesis and release, as well as Reactive Oxygen Species (ROS) generation. Many of these functions are positively coupled with cytoplasmic pH (pHi) and/or phagosomal pH (pHp) modification; an increase in pHi represents an important signal for cytokine and chemokine release, whereas a decrease in pHp can induce an efficient antigen presentation. However, the relationship between pHi and ROS generation is not well understood. In immune cells two main transport systems have been shown to regulate pHi: the Na+/H+ Exchanger (NHE) and the plasmalemmal V-type H+ ATPase. NHE is a family of proteins which exchange Na+ for H+ according to their concentration gradients in an electroneutral manner. The exchanger also plays a key role in several other cellular functions including proliferation, differentiation, apoptosis, migration, and cytoskeletal organization. Since not much is known on the relationship between NHE and immunity, this review outlines the contribution of NHE to different aspects of innate and adaptive immune responses such as phagosomal acidification, NADPH oxidase activation and ROS generation, cytokine and chemokine release as well as T cell apoptosis. The possibility that several pro-inflammatory diseases may be modulated by NHE activity is evaluated.
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Affiliation(s)
- Paolo De Vito
- Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy.
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Hubert A, Cauliez B, Chedeville A, Husson A, Lavoinne A. Osmotic stress, a proinflammatory signal in Caco-2 cells. Biochimie 2005; 86:533-41. [PMID: 15388230 DOI: 10.1016/j.biochi.2004.07.009] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2003] [Accepted: 07/16/2004] [Indexed: 11/17/2022]
Abstract
Hyper- (450 mOsm/l) and hypoosmotic exposure (150 mOsm/l) of Caco-2 cells, a human intestinal epithelial cell line, induced a twofold- and a fivefold increase in the production of IL-8, a constitutively expressed cytokine, respectively. This was observed both in the presence or in the absence of added proinflammatory cytokines and the stimulatory effect of osmotic stress was additive to that induced by the cytokines. Thus, IL-8 production appeared minimal around isoosmolarity, i.e. 300 mOsm/l. Concerning the signalling pathway involved, specific inhibition of p38- or p42/44 MAP kinases decreased the IL-8 production by about 30% independently of the osmotic condition used. Inhibition of c-jun-NH2-terminal kinase (JNK) by using both dicoumarol and SP600125 totally inhibited the stimulatory effect of hypoosmolarity. Moreover, hypoosmolarity induced an about threefold increase in JNK activity demonstrating that JNK was specifically involved in the effect of hypoosmolarity on IL-8 production. This is not the case for hyperosmolarity. Such an effect of osmotic stress was not restricted to IL-8, but was also observed on the production of IL-6, a non-constitutively expressed cytokine. Again, IL-6 production appeared minimal in isoosmotic condition. Taken together, these results demonstrate that osmotic stress is a proinflammatory signal in Caco-2 cells and suggest that an osmosensor might specifically exist in intestinal epithelial cells.
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Affiliation(s)
- Aurélie Hubert
- ADEN, EA-3234, IFR-23, UFR Médecine-Pharmacie de Rouen, 22, bd Gambetta, 76183 Rouen, France
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Ohkubo R, Tomita M, Hotta Y, Nagira M, Hayashi M. Comparative Study of Flux of FITC-labeled Dextran 4000 on Normal (iso)- and Hyper-osmolarity in Basal Side in Caco-2 Cell Monolayers. Drug Metab Pharmacokinet 2003; 18:404-8. [PMID: 15618761 DOI: 10.2133/dmpk.18.404] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We have shown previously that the flux of fluorescein isothiocyanate dextran 4000 (FD-4) is transported across the Caco-2 cell monolayers in a polarized fashion favoring the basal to apical direction under normal conditions (i.e., isotonic solution in basal side). Furthermore, FD-4 transport may occur via a process that included a certain degree of substrate specificity for polysaccharide and transcytosis. In the present study, we compared the flux of FD-4 in the basal to apical direction (efflux) and the apical to basal direction (influx) in stress conditions (i.e., hyperosmolarity in basal side) to those in normal conditions (i.e., iso-osmolarity in basal side). The efflux of FD-4 was increased by hyperosmolarity in basal side, but the influx was decreased when compared with normal conditions. Neither dextran 10, 000 nor colchicine inhibited the efflux of FD-4 in hyperosmolarity conditions. The inhibition of efflux of FD-4 was observed not by S-nitroso-N-acetylpenicillamine but by sodium nitroprusside and sodium ferrocyanide. These results collectively suggest that hyperosmolarity in basal side accelerates the efflux of FD-4 across the transcellular route but not across the paracellular route in Caco-2 cell monolayers. And it is indicated that cyanide rather than nitric oxide is involved in dysfunction of the FD-4 efflux system irrespective of conditions such as normal osmolarity or hyperosmolarity.
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Affiliation(s)
- Rie Ohkubo
- Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan
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Németh ZH, Deitch EA, Szabó C, Haskó G. Hyperosmotic stress induces nuclear factor-kappaB activation and interleukin-8 production in human intestinal epithelial cells. THE AMERICAN JOURNAL OF PATHOLOGY 2002; 161:987-96. [PMID: 12213727 PMCID: PMC1867255 DOI: 10.1016/s0002-9440(10)64259-9] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Inflammatory bowel disease of the colon is associated with a high osmolarity of colonic contents. We hypothesized that this hyperosmolarity may contribute to colonic inflammation by stimulating the proinflammatory activity of intestinal epithelial cells (IECs). The human IEC lines HT-29 and Caco-2 were used to study the effect of hyperosmolarity on the IEC inflammatory response. Exposure of IECs to hyperosmolarity triggered expression of the proinflammatory chemokine interleukin (IL)-8 both at the secreted protein and mRNA levels. In addition, hyperosmotic stimulation induced the release of another chemokine, GRO-alpha. These effects were because of activation of the transcription factor, nuclear factor (NF)-kappaB, because hyperosmolarity stimulated both NF-kappaB DNA binding and NF-kappaB-dependent transcriptional activity. Hyperosmolarity activated both p38 and p42/44 mitogen-activated protein kinases, which effect contributed to hyperosmolarity-stimulated IL-8 production, because p38 and p42/44 inhibition prevented the hyperosmolarity-induced increase in IL-8 production. In addition, the proinflammatory effects of hyperosmolarity were, in a large part, mediated by activation of Na(+)/H(+) exchangers, because selective blockade of Na(+)/H(+) exchangers prevented the hyperosmolarity-induced IEC inflammatory response. In summary, hyperosmolarity stimulates IEC IL-8 production, which effect may contribute to the maintenance of inflammation in inflammatory bowel disease.
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Affiliation(s)
- Zoltán H Németh
- Department of Surgery, University of Medicine and Dentistry-New Jersey Medical School, Newark 07103, USA
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Fonti R, Latella G, Caprilli R, Frieri G, Marcheggiano A, Sambuy Y. Carbonic anhydrase I reduction in colonic mucosa of patients with active ulcerative colitis. Dig Dis Sci 1998; 43:2086-92. [PMID: 9753277 DOI: 10.1023/a:1018819600645] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Ulcerative colitis (UC) is associated with low intracolonic pH and unbalanced transmucosal ionic exchanges. Along the gastrointestinal tract carbonic anhydrase isoenzyme I (CA-I) is specifically expressed in colon epithelium and is involved in mucosal control of ion, fluid, and acid-base balance. Since altered CA-I expression may play some role in UC, CA-I was measured at the mRNA and protein level and carbonic anhydrase (CA) enzyme activity was determined in colon biopsies of 14 UC patients (6 remission, 4 mild, 4 moderate UC) and of 12 healthy subjects. Patients with mild or moderate UC showed a significant reduction of CA-I mRNA and protein and of total CA activity in the inflamed mucosa compared to controls. Patients with UC in remission showed a pattern of CA-I expression and CA activity similar to controls. This is the first report showing a reduction in the expression of CA-I in active UC.
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Affiliation(s)
- R Fonti
- Cattedra di Gastroenterologia, Università de L'Aquila, Italy
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Gaginella TS, Kachur JF, Tamai H, Keshavarzian A. Reactive oxygen and nitrogen metabolites as mediators of secretory diarrhea. Gastroenterology 1995; 109:2019-28. [PMID: 7498670 DOI: 10.1016/0016-5085(95)90772-6] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Affiliation(s)
- T S Gaginella
- Searle Research & Development, Skokie, Illinois, USA
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Frieri G, Ligas E, Perugini B, Onori L, Marcheggiano A, Caprilli R. Ulcerative colitis: rectal dilations in a patient with refractory diarrhea. Report of a case. Dis Colon Rectum 1995; 38:545-9. [PMID: 7736888 DOI: 10.1007/bf02148858] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
UNLABELLED A 45-year-old man with an eight-year history of ulcerative colitis was evaluated for severe, nonbloody diarrhea. Symptoms, which began two years earlier, were characterized by 15 bowel movements per day, accompanied by urgency and incontinence. A reduced rectal compliance was measured at manometry. All conventional treatments were not able to modify the symptoms despite improvement of inflammatory colonic lesions. PURPOSE The aim was to reduce bowel movements and incontinence by increasing rectal compliance. METHODS Gradual pneumatic dilations of the rectum were performed three times per week for three weeks. RESULTS The patient's diarrhea improved dramatically, decreasing in frequency from approximately 15 to only 3 bowel movements per day, while contemporaneously increasing rectal compliance. Such effect, still evident 15 months after discontinuation of dilation, was probably obtained by improvement of viscoelastic features of the intestinal wall. CONCLUSIONS Rectal pneumatic dilation may be a successful attempt in some forms of intractable diarrhea in long-lasting ulcerative colitis.
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Affiliation(s)
- G Frieri
- Department of Internal Medicine and Public Health, University of L'Aquila, Italy
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49
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Agarwal R, Afzalpurkar R, Fordtran JS. Pathophysiology of potassium absorption and secretion by the human intestine. Gastroenterology 1994; 107:548-71. [PMID: 8039632 DOI: 10.1016/0016-5085(94)90184-8] [Citation(s) in RCA: 126] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
When normal people ingest 90 mEq/day of K+ in their diet, they absorb about 90% of intake (81 mEq) and excrete an equivalent amount of K+ in the urine. Normal fecal K+ excretion averages about 9 mEq/day. The vast majority of intestinal K+ absorption occurs in the small intestine; the contribution of the normal colon to net K+ absorption and secretion is trivial. K+ is absorbed or secreted mainly by passive mechanisms; the rectum and perhaps the sigmoid colon have the capacity to actively secrete K+, but the quantitative and physiological significance of this active secretion is uncertain. Hyperaldosteronism increases fecal K+ excretion by about 3 mEq/day in people with otherwise normal intestinal tracts. Cation exchange resin by mouth can increase fecal K+ excretion to 40 mEq/day. The absorptive mechanisms of K+ are not disturbed by diarrhea per se, but fecal K+ losses are increased in diarrheal diseases by unabsorbed anions (which obligate K+), by electrochemical gradients secondary to active chloride secretion, and probably by secondary hyperaldosteronism. In diarrhea, total body K+ can be reduced by two mechanisms: loss of muscle mass because of malnutrition and reduced net absorption of K+; only the latter causes hypokalemia. Balance studies in patients with diarrhea are exceedingly rare, but available data emphasize an important role for dietary K+ intake, renal K+ excretion, and fecal K+ losses in determining whether or not a patient develops hypokalemia. The paradoxical negative K+ balance induced by ureterosigmoid anastomosis is described. The concept that fecal K+ excretion is markedly elevated in patients with uremia as an intestinal adaptation to prevent hyperkalemia is analyzed; we conclude that the data do not convincingly show the existence of a major intestinal adaptive response to chronic renal failure.
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Affiliation(s)
- R Agarwal
- Department of Internal Medicine, Baylor University Medical Center, Dallas, Texas
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Brevinge H, Bosaeus I, Philipson BM, Kewenter J. Sodium and potassium excretion before and after conversion from conventional to reservoir ileostomy. Int J Colorectal Dis 1992; 7:148-54. [PMID: 1402313 DOI: 10.1007/bf00360356] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Sodium and potassium in the ileostomy output and urine were determined in 28 patients with ulcerative colitis on a free diet and in eight patients on a defined constant diet, before and after conversion from a conventional ileostomy (CI) to a continent reservoir ileostomy (RI). Feces and urine were collected both in the hospital and at home. Patients with CI on free diet had a median intestinal loss of 62 mmol sodium and those with RI 74 mmol/24 h collected in the hospital (p < 0.05). The figures for at home was 79 and 81 mmol/24 respectively, and were larger than in the hospital (p < 0.01). Sodium loss in the urine (U-Na) and the intake of sodium did not change significantly after conversion. Patients with a low U-Na before conversion also had a low U-Na after, in a few almost nil, implying a need for increased intake of sodium. Patients with a CI and low urinary output of sodium should be carefully studied with respect to their sodium balance before accepting them for conversion to RI. The ileostomy output of potassium increased after conversion (4.3 vs. 6.8 mmol/24 h; p < 0.01) in the hospital (5.3 vs 7.1 mmol/24 h; p < 0.01) at home. Patients on a defined constant diet before and after conversion did not show any significant differences in absorption of sodium, potassium, magnesium or calcium after conversion, but did show a reduced dry weight of the ileostomy output, indicating an increased degradation of intestinal contents in RI patients.(ABSTRACT TRUNCATED AT 250 WORDS)
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Affiliation(s)
- H Brevinge
- Department of Surgery, University of Göteborg, Sweden
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