Gastrointestinal Physiology and Pathophysiology


Chapter 1.1

Gastrointestinal Physiology and Pathophysiology



N.C. Zachos    Hopkins Conte Digestive Disease Basic and Translational Research Core Center, Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, MD, United States


Abstract


Rotaviruses, noroviruses, astroviruses, and enteric adenoviruses are the four major viral causes of acute gastroenteritis, which is a major public health concern and a common cause of morbidity worldwide and mortality in low income settings. Although the intestinal epithelium serves as a protective barrier, enteric viral pathogens disrupt normal intestinal homeostasis, resulting in excessive nutrient and water loss. However, the cellular and molecular mechanisms responsible for viral diarrhea are not fully understood. While significant advances in viral diarrheal disease research have been accomplished, most of our understanding of the pathogenesis of gastroenteritis has come from studies of bacterial pathogens. The goal of this chapter is to describe the current understanding of normal intestinal physiology and compare the molecular mechanisms that are altered in diarrheal diseases induced by bacterial or viral pathogens.



Keywords


gut physiology

gut pathophysiology

fluid balance

intestinal absorption

sugar transport

anion secretion

intestinal enteroids


1. Introduction


The intestinal epithelium constantly manages the complex regulation of paracellular and transcellular transport of water, electrolytes, and small solutes to promote nutrient absorption and secretion of various compounds (electrolytes, methyl sulfides, benzopyrrole derivatives a.o.) while preventing excess fluid loss. The balance of absorptive and secretory processes is tightly regulated to manage the nearly 9 L/day of fluid in the intestinal lumen, of which nearly 98% are absorbed by the intestinal epithelium. These functions occur through the coordinated interplay of polarized columnar epithelial cells that are aligned in a continuous monolayer contoured by villi and crypts, which in addition to epithelial microvilli, enhance the plain surface area by greater than 600-fold (Montrose et al., 1999). The villous epithelium is predominantly comprised of mature enterocytes along with mucus-secreting Goblet cells, hormone producing entero-endocrine cells, and tuft cells while the crypt epithelium is mostly composed of immature enterocytes with Paneth and stem cells located at the crypt base (Barker et al., 2008Cheng and Leblond, 1974). Pioneering work by the laboratory of Hans Clevers has identified Lgr5 as the molecular marker for the constantly dividing intestinal stem cells that differentiate into all intestinal epithelial cell types (Sato et al., 2011a). Intestinal stem cells confer segment-specific functions such that the proximal small intestine absorbs carbohydrates and fatty acids while the ileum absorbs bile acids and vitamin B12 (Middendorp et al., 2014). Nutrient and salt uptake by the intestinal epithelium generates an osmotic gradient that drives the active (via apical ion/solute cotransporters) and/or passive (ie, paracellular) absorption of most of the water exposed per day to the intestine. In the absence of nutrients, small intestinal and proximal colonic fluid absorption occurs through the electroneutral coupled absorption of Na+ and Cl while electrogenic absorption of Na+ predominates in the distal colon (Montrose et al., 1999).

Across all age groups, diarrhea is one of the top five causes of death worldwide; while in children less than 5 years old, diarrhea is the second leading cause of death. Diarrhea etiology includes bacteria, viruses, parasites, toxins, or drugs. Acute viral gastroenteritis remains a major public health concern and a common cause of morbidity and mortality worldwide. Rotaviruses, caliciviruses (particularly norovirus), astroviruses, and enteric adenoviruses are the four predominant causes of viral gastroenteritis, inducing symptoms ranging from self-limiting watery diarrhea that resolves within 7 days to severe dehydration (with complications including cerebral edema, hypovolemic shock, renal failure) and death. Two recently developed rotavirus vaccines (RotaTeq® RV5 and Rotarix® RV1) have reduced rotavirus disease in developed countries but are much less effective in low income settings (Babji and Kang, 2012Tate et al., 2013). The recent Global Enteric Multicenter Study (GEMS) confirmed that rotavirus is the leading cause of infant diarrhea among the more than 20,000 children studied in seven sites across Asia and Africa (Kotloff et al., 2013). Although Oral Rehydration Solution (ORS) remains the only safe, effective, and low cost therapeutic option to prevent life-threatening dehydration due to acute severe diarrheal diseases (Binder et al., 2014), the GEMS study reported that each episode of severe diarrhea in children increased the risk of delayed physical and intellectual development as well as increased mortality by 8.5-fold (Kotloff et al., 2013). Therefore, there remains a great need to develop additional therapies to treat severe diarrheal diseases.

Diarrhea often involves the dysregulation of absorptive processes and/or loss of epithelial barrier integrity, resulting in nutrient malabsorption and/or activated secretion of fluid into the intestinal lumen. In addition to the contribution of the enteric nervous system to diarrhea, roles for intestinal microbiota and epithelial immunity have been described (Moens and Veldhoen, 2012). While numerous studies of bacterial pathogens have improved our understanding of the pathogenesis of gastroenteritis, other studies suggest that viral diarrhea occurs by distinctly different mechanisms (Lorrot and Vasseur, 2007). This chapter will describe the current understanding of normal human intestinal physiology and compare the molecular mechanisms that are dysregulated by enteric bacterial and viral pathogens in the various model systems studied to date.

2. Normal intestinal physiology


Under normal digestive conditions, the intestine is exposed to ∼9 L of fluids daily. The ability of the intestine to efficiently absorb dietary water, electrolytes and nutrients is defined by specific functional processes that occur along the horizontal (ie, proximal to distal intestinal segments) and vertical (ie, crypt/villus) axes. While the entire intestine is capable of absorbing water and salts, uptake of carbohydrates, peptides, amino acids, minerals, vitamins, long/short chain fatty acids, and bile acids occurs in distinct intestinal segments that uniquely express the transport proteins to facilitate absorption (Fig. 1.1.1). These proteins are asymmetrically expressed in polarized epithelial cells to perform vectorial transport across the intestinal epithelium resulting in net nutrient and water absorption (Fig. 1.1.2). The intestinal architecture enhances absorption through the increased surface area generated by the villus/crypt axis as well as by the densely packed microvilli of mature enterocytes.

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Figure 1.1.1 Segment-specific functions in the human small intestine and colon.
Colored boxes correspond to similar colored intestinal segments and describe the specific absorptive/secretory function of the respective intestinal segment. “+” indicates amount of fluid, including gastric, pancreatic, and intestinal secretions, secreted into intestine daily. SCFA, short chain fatty acids; NDO, nondigestable oligosaccharides.

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Figure 1.1.2 Normal small intestinal nutrient and electrolyte absorption.
The basolateral Na+/K+-ATPase drives the Na+ gradient across the apical and basolateral membranes. Electroneutral NaCl absorption takes place through the coupled regulation of NHE3 and DRA. Peptides and amino acids are transported by the apical PEPT1 and Na+-dependent amino acid transporters (eg, B0AT1), respectively. Intracellular peptidases complete breakdown of absorbed peptides into amino acids that are exported from the epithelium through basolateral amino acid transporters (eg, ASCT1). The dietary sugars, glucose, galactose and fructose are absorbed by apical transporters SGLT-1 and GLUT5. SGLT-1 absorbs glucose in a Na+-dependent manner while GLUT5 absorbs fructose through facilitative transport. GLUT2 normally resides on the basolateral membrane in between meals to complete transcellular movement of dietary sugars to the portal circulation. However, after a carbohydrate-rich meal, GLUT2 translocates to the apical membrane to handle excess luminal concentrations of glucose that are beyond the capacity of SGLT-1 to absorb. Apical and basolateral potassium channels maintain cellular electrical neutrality during active transport. Carbonic anhydrase maintains physiological pH and fluid balance. nAA, neutral amino acids; Glu, glucose; Gal, galactose; Fruc, fructose; pep, peptide; TJ, tight junction. Black denotes Brush border. Red denotes basolateral membrane.

The intestinal epithelium serves as a barrier separating luminal dietary and microbial content from the bloodstream. A single monolayer of polarized columnar epithelial cells is connected by tight junctions that segregate the epithelial apical and basolateral membranes. Tight junctions are formed and maintained by dynamic multiprotein complexes that regulate the paracellular movement of water, ions, and small molecules. Occludin and the claudin family of proteins regulate paracellular movement of ions and small molecules through selectivity based on electrical conductance, size, and charge (Anderson and Van Itallie, 2006). Of these, electrical conductance is the most variable (>fivefold difference) between the “leaky” and “tight” epithelium of the duodenum and distal colon, respectively. The expression of different claudin isoforms varies along the horizontal and vertical axes conferring intestinal segment-specific regulation of paracellular transport (Holmes et al., 2006). Movement through tight junctions is a passive process generated by chemical and osmotic gradients that result from active transcellular transport during the postprandial state. Tight junctions regulate the rate of paracellular fluid flow through the opening and closing of the claudin pores (Anderson and Van Itallie, 2006).

The intestinal epithelium is comprised of the major epithelial cell types: enterocytes, entero-endocrine, goblet, tuft and Paneth cells. The leucine-rich G-protein coupled receptor 5, Lgr5, has been established as the molecular marker for the actively dividing intestinal stem cells that give rise to all epithelial cell types (Sato et al., 2011a). In the small intestine, Lgr5+ stem cells reside at the base of the crypt, interspersed among Paneth cells, and undergo asymmetric division producing immature enterocytes which comprise the crypt transit amplifying zone. These enterocytes terminally differentiate into the other epithelial cell types, including mature enterocytes and secretory cell lineages, as they migrate towards the villus tips (Barker et al., 2008). The life cycle of the small intestinal epithelium is about 5–7 days; while Paneth and stem cells turn over approximately every 30 days. This rapid renewal is necessary to handle the harsh luminal environment, and thus the intestinal epithelium is considered the most regenerative region in the body.

In addition to promoting nutrient digestion and absorption, the intestinal epithelial barrier protects the body from luminal pathogens and their secreted products. The intestinal epithelium is exposed to the largest population of microbial species (ranging from <104/mL in the small intestine to >1015/mL in the colon) and serves as the first line of defense against harmful microbial pathogens. Goblet cells constantly secrete highly glycosylated proteins, called mucins (MUC), to produce a mucus layer that serves as a physical barrier, preventing direct interaction of commensal microbes and enteric pathogens with the host epithelium. The mucus layer is a complex organization of transmembrane and gel-forming (ie, secreted) MUC molecules that develop a tightly adhered inner layer and loose outer layer, respectively (Johansson et al., 2013). The small intestine has a single, loose unattached mucus layer comprised exclusively of the gel forming MUC2 while the colon has a two-layered system in which MUC2 serves as the foundation for both layers. The mucus of the small intestine serves to provide a nearly sterile environment through hourly turnover of the mucus layer and antimicrobial peptide secretion by Paneth cells (Bevins et al., 1999). The constant turnover occurs through regulated anion secretion during digestion and between meals. The inner mucus layer of the colon is constantly regenerated and maintained by surface goblet cells. The outer layer of the colonic mucus originates from the inner layer which becomes loose and unattached allowing commensal bacteria to reside there (Johansson et al., 2013). While the intestinal mucus is the first line of defense against enteric pathogens, enteric viruses evade this protective barrier by mechanisms requiring further study. A single report suggested that small intestinal and colonic mucins (colonic mucus had stronger effect) can prevent binding and entry of rotavirus and thus viral replication (Chen et al., 1993). Further studies are required to understand the mechanisms responsible for this effect.

2.1. Fluid Balance in the Intestine


The average dietary intake of fluid, which includes water, electrolytes, and nutrients, is ∼2 L; however, due to the secretion of fluids necessary for normal digestion which include saliva, bile, as well as gastric, pancreatic, and intestinal secretions, the total volume of fluid in contact with the intestinal epithelium is ∼9 L/day. Together, the small intestine (∼7 L) and the colon (∼2 L) have the capacity to absorb more than 98% of this fluid load, leaving ∼200 mL for excretion in stool (Fig. 1.1.1). Of that absorbed by the small intestine, ∼5 L is taken up by the duodenum and jejunum while the ileum is responsible for the remaining ∼2 L. Although the small intestinal handles a majority of the fluid absorption, the colon has the capacity to absorb up to 6 L daily (Montrose et al., 1999).

Since the fluid secreted into the intestinal lumen is comprised of gastric, pancreatic and intestinal juices, increased fluid secretion facilitates the spreading of enzymes for normal digestive processes. In addition, increased fluid secretion protects the intestinal mucosa from potential damage as well as allowing stool to pass through the gut. While many studies have elucidated the molecular mechanisms involved in increased water secretion during enteric infections (Hodges and Gill, 2010Morris and Estes, 2001Viswanathan et al., 2009), a basic understanding of how water is secreted under basal or normal digestive conditions is still lacking. Moreover, a definitive path (ie, paracellular vs transcellular) for water absorption/secretion in the human intestine has yet to be determined.

The aquaporin (AQP) family of proteins is comprised of 13 isoforms that are categorized into two groups: the AQPs that are only permeable to water, and the aquaglyceroporins that are permeable to water, glycerol, urea, and other small solutes (Agre and Kozono, 2003). AQPs are responsible for osmotic pressure-driven movement of water from the lumen by the intestinal epithelium; however, whether any AQPs participate in active water transport against osmotic gradients remains to be determined. AQP isoforms 1, 3, 7, 10, and 11 are expressed in epithelial cells of both the human small intestine and colon while AQPs 4 and 8 are colonic (Laforenza, 2012). In addition, AQP1 is expressed in the capillary endothelium of the small intestine and may serve as the pathway for water movement from the intestinal mucosa into the circulation. However, detailed characterization of the segment-specific expression and localization of each AQP isoform in the human intestine is incomplete. Understanding the roles of each AQP in water transport under normal digestive conditions and in diarrheal disease in humans is complicated due to conflicting data from animal models in which AQP−/− mice do not exhibit abnormal intestinal phenotypes (Agre and Kozono, 2003). The hypothesis that aquaporins are necessary for normal water transport in humans is supported by correlative data from inflammatory bowel disease, celiac disease, and trichohepatoenteric syndrome patients that have decreased expression of one or more AQP isoforms suggesting that lack of AQPs results in decreased luminal water absorption (Guttman and Finlay, 2008Hartley et al., 2010). Rodent studies of bacterial pathogenesis observed that decreased AQP activity is due to post translational modifications and to protein trafficking induced by bacterial virulence factors (Guttman et al., 2007). Infection of mice by the attaching and effacing bacterial pathogen, Citrobacter rodentium, resulted in mislocalization of AQPs 2 and 3 from the apical membrane to intracellular vesicles. This effect was due to the activity of the bacterial virulence factors EspF and EspG that are injected into host cells by the bacterial type III secretion system. Normal apical membrane expression of AQPs 2 and 3 was restored after C. rodentium was cleared from mice (Guttman et al., 2007). One recent report has demonstrated that RV reduces the expression of AQPs 1, 4, and 8 in a mouse model of RV diarrhea (Cao et al., 2014). The roles of AQPs in normal intestinal water movement and in enteric viral pathogenesis remain to be determined.

2.2. Intestinal Absorption


2.2.1. Sugar Transport


Intestinal absorption of the main dietary sugars (ie, glucose, galactose, and fructose) relies on the coordinated function of transporters at the apical and basolateral membranes of enterocytes (Shirazi-Beechey et al., 2011). In between meals, low luminal levels of glucose are absorbed at the apical membrane of enterocytes by the Na+-dependent glucose transporter, SGLT1, while fructose is absorbed by GLUT5, a facilitative transporter (Mueckler, 1994). SGLT1 has a stoichiometry of 2 Na+:1 glucose during each transport cycle. Na+ uptake by SGLT1 is removed from the cell by the basolateral Na+/K+ ATPase, and the resulting electrochemical gradient increases paracellular transport of Cl from the lumen (Fig. 1.1.2). In addition, activation of SGLT1 leads to increased expression and activity of NHE3 at the brush border (BB) (Hu et al., 2006Lin et al., 2011). Increased NHE3 activity results in intracellular alkalinization which stimulates BB Cl/HCO3 exchange activity and apical Cl absorption.

The predominantly basolaterally expressed glucose/fructose transporter, GLUT2, provides an exit pathway for glucose and fructose into the bloodstream (Roder et al., 2014). GLUT2 is able to process high sugar concentrations efficiently due to its high maximum rate (Vmax) of transporting high concentrations of glucose and its high affinity for binding glucose (Michaelis–Menten constant; Km) compared to SGLT1, which has lower transport rate and affinity for glucose (Raja et al., 2012). SGLT1, GLUT5, and GLUT2 are expressed in the duodenum and jejunum, and at lower levels in the ileum. Upon digestion of carbohydrate-rich meals that produce luminal glucose concentrations above SGLT1 saturating concentrations, unsaturated GLUT2 rapidly (within minutes) translocates to the apical membrane of enterocytes, and is also rapidly retracted back to the basolateral membrane when luminal glucose levels decrease (Gouyon et al., 2003). Different signaling mechanisms have been shown to trigger insertion of GLUT2 into the apical membrane, including high luminal sugar concentrations, stress, corticoids, and enteroendocrine hormones (Au et al., 2002Chaudhry et al., 2012Mace et al., 2007Shepherd et al., 2004). Under normal physiological conditions, insulin triggers GLUT2 internalization, which then limits any increase in serum glucose levels (Tobin et al., 2008).

2.2.2. Amino Acid and Peptide Transport


The small intestine is the major region responsible for amino acid and peptide absorption (Fig. 1.1.1). Dietary proteins are digested to smaller peptide fragments and free amino acids by gastric, pancreatic, and intestinal enzymes. The BBs of differentiated intestinal epithelial cells express additional digestive enzymes that further hydrolyze oligopeptides to smaller di- and tri-peptides, including aminopeptidase N (APN), carboxypeptidase, and dipeptidylpeptidase IV (DPPIV). All hydrolyzed di- and tri-peptides are absorbed by the BB electrogenic protein/peptide symporter, PEPT1; however, PEPT1 does not transport free amino acids or peptides larger than three amino acids (Daniel, 2004). PEPT1 activity is functionally linked to NHE3 activity through recycling of protons (Kennedy et al., 2002). NHE3 extrudes intracellular protons for PEPT1 symport that in turn lowers intracellular pH and activates NHE3 facilitating Na+ absorption. Acute second messenger elevation leads to decreased PEPT1 activity similar to that described for NHE3 (Daniel, 2004). Insulin, leptin, growth hormone, and thyroid hormone have all been demonstrated to increase abundance and activity of PEPT1 (Daniel, 2004). Absorbed di- and tri-peptides are further digested by intracellular proteases to free amino acids which are transported to the portal circulation by basolaterally expressed amino acid transporters. Amino acid transporters are expressed at both the apical and basolateral membranes of small intestinal epithelial cells and are characterized by their dependence on a Na+ gradient for transport activity as well as by the class of amino acids they transport (ie, neutral, anionic, cationic, or imino acids) (Broer, 2008).

2.2.3. Electroneutral NaCl Absorption


Electroneutral NaCl absorption occurs throughout the mammalian intestine (except the distal colon) and accounts for most intestinal Na+ absorption in the period between meals (Fig. 1.1.1). This process contributes more to Na+ absorption in the ileum and proximal colon post prandially than in the proximal small intestine, where amino acid, peptide, and carbohydrate symporters are linked to Na+ absorption (Maher et al., 1996 1997Zachos et al., 2005). Electroneutral NaCl absorption is both up- and downregulated as part of digestion, appearing to be inhibited initially during and after eating (for spreading digestive enzymes) and then stimulated later in digestion (for fluid absorption) (Donowitz and Tse, 2000). This regulation occurs in response to eating via the complex neural/paracrine/endocrine changes in the intestine. In diarrheal diseases, electroneutral NaCl absorption is inhibited while electrogenic Cl secretion is stimulated. These events are responsible for the major loss of water and electrolytes in diarrhea (Donowitz and Tse, 2000). Electroneutral NaCl absorption occurs through the functional linkage of a Na+ absorptive transporter and a Cl/HCO3 exchanger. Regulation of the BB Na+/H+ exchanger, NHE3 (SLC9a3), accounts for most of the recognized digestive changes in electroneutral NaCl absorption, as well as most of the changes in Na+ absorption that occur in diarrheal diseases (Zachos et al., 2005). The BB Cl/HCO3 exchangers, downregulated in adenoma (DRA; SLC26a3) and putative anion transporter (PAT1; SLC26a6), are functionally linked to NHE3 activity by intracellular carbonic anhydrase which provides the H+ and HCO3 for NHE3 and DRA/PAT1 functions, respectively (Melvin et al., 1999Simpson et al., 2010Walker et al., 2008). These Cl/HCO3 exchangers function in anion secretion as well as Cl absorption. DRA is predominantly expressed in the duodenum and colon while PAT1 is expressed in the jejunum and ileum. Elevation of second messengers (ie, cAMP, cGMP, Ca2+) has been shown to acutely regulate NaCl absorption by inhibiting or stimulating the function of NHE3 and/or DRA/PAT1 (Chen et al., 2015Musch et al., 2009Xia et al., 2014). In the distal colon, ∼50% of luminal Na+ absorption is electrogenic and occurs through the regulation of the apical epithelial Na+ channel, ENaC. Examples of regulation include aldosterone (acting via signal transduction) and CFTR, which directly binds and modulates ENaC activity (Wagner et al., 2001).

2.2.4. Anion Secretion


Chloride is the major anion responsible for generating the osmotic gradient necessary to drive the active transport of water across the intestinal epithelium and into the lumen (Fig. 1.1.3). HCO3 can also increase water transport; however, to a lesser extent. In intestinal epithelial cells, increased apical Cl channel activity and fluid secretion requires the contribution of basolateral transport processes including Na+/K+/2Cl symporter (NKCC1), Na+/K+ ATPase, and K+ channels (Kunzelmann et al., 2001Payne et al., 1995Tabcharani et al., 1991). These three processes collectively contribute to increasing the intracellular concentration of Cl stimulating the efflux of Cl down its electrochemical gradient through apical chloride channels. NKCC1-mediated transport of Cl across the basolateral membrane is driven by the Na+ gradient (via efflux) established by the Na+/K+ ATPase, which exchanges 3 Na+ (efflux) for 2 K+ (influx) (Montrose et al., 1999). K+ ions transported into the cell by both NKCC1 and Na+/K+ ATPase are leaked out by basolateral K+ channels (eg, KCNQ1/KCNE3) to maintain cellular electroneutrality to compensate for the loss of Cl. K+ efflux results in membrane hyperpolarization and is the main driving force for Cl secretion (Heitzmann and Warth, 2008). The loss of Cl and K+ establishes the negative charge and the accumulation of luminal Cl subsequently drives the paracellular transport of Na+ through the tight junctions and into the intestinal lumen (Field, 2003). This mucosal to luminal transport of NaCl facilitates the osmotic flow of water and thus net secretion.

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Figure 1.1.3 Active fluid secretion is mediated through inhibition of apical NaCl absorption and increased anion secretion.
Second messenger elevation (eg, cAMP or Ca2+) inhibits NHE3 and apical Cl/HCO3 exchange (via DRA/PAT1) while stimulating Cl secretion through apical Cl channels, CFTR, CaCC, or ClC-2. The basolateral Na+/K+/2Cl cotransporter provides the Cl gradient for secretion. Elevated levels of cAMP and Ca2+ can both stimulate CFTR activity by increasing apical membrane expression through increased trafficking as well as open channel probability. The molecular identity of the CaCC involved in intestinal fluid secretion remains to be determined.

In the intestine, the apical Cl channels that contribute to fluid secretion include: Cystic Fibrosis Transmembrane Regulator (CFTR), Ca2+ activated chloride channels (CaCC), and chloride channel 2 (ClC2). CFTR is the major intestinal apical Cl channel, which is expressed in enterocytes from the proximal small intestine to the distal colon. In intestinal enterocytes, elevation of cAMP increases CFTR activity (ie, open channel probability) through: (1) direct phosphorylation of its regulatory domain leading to ATP hydrolysis, or (2) increased trafficking to the apical membrane (Tabcharani et al., 1991). In addition to cAMP, elevation of intracellular Ca2+ and cGMP levels increase CFTR activity. The contribution of CFTR to intestinal secretion is emphasized in cystic fibrosis (CF) patients who suffer from chronic constipation (among other symptoms). Small molecule CFTR potentiators are currently being developed to increase CFTR activity through increased BB insertion based on a CF patient’s genotype (Dekkers et al., 2013). Secretory diarrheas induced by the enterotoxins of Vibrio cholerae (ie, cholera toxin; CTx) and enterotoxigenic Escherichia coli (ie, heat stable enterotoxin; STa) involve stimulation of CFTR activity through elevated cAMP and cGMP levels, respectively. Recently, three classes of highly potent (ie, low nM) CFTR inhibitors have been developed and may serve as potential antidiarrheal agents: (1) the thiazolidinone CFTRinh-172 binds to cytoplasmic side of CFTR and stabilizes its closed conformational state; (2) the PPQ/BPO compounds (R-BPO-27: IC50 ∼4 nM) also target the cytoplasmic side of CFTR; and (3) the glycine hydrazide, GlyH-101, inhibits CFTR by binding the channel pore from the extracellular side (Thiagarajah et al., 2014). Both CFTRinh-172 and GlyH-101 have demonstrated antisecretory effects in rodent diarrheal models while R-BPO-27 has only been tested in models of polycystic kidney disease. Natural products including the plant extract SB-300 and a Thai herbal remedy have been shown to also effectively inhibit CFTR activity in models of diarrheal disease (Fischer et al., 2004Tradtrantip et al., 2014).

Luminal purinergic or basolateral adrenergic and cholinergic signaling lead to increases in intracellular Ca2+ that evokes a transient Cl secretory response. Studies using intestinal epithelial cell lines have observed that the Cl secretory response to Ca2+ is not as strong or sustained when compared to cAMP stimulated Cl secretion. Moreover, the effects of Ca2+ and cAMP on Cl secretion are synergistic, suggesting that another Ca2+-dependent, cAMP-independent Cl channel mediates this secretion; however, the molecular identity of the apical CaCC responsible is not clear (Thiagarajah et al., 2015). The anoctamins (particularly TMEM16A) and bestrophins have been suggested as candidate CaCCs; however, studies have indicated that they do not contribute to intestinal Cl secretion. Moreover, CFTR knock-out (KO) mice do not exhibit an intestinal Cl secretory response to cAMP or Ca2+ agonists (Thiagarajah et al., 2015). An alternate hypothesis suggests that Ca2+-mediated Cl secretion occurs due to activation of basolateral K+ channels which would indirectly stimulate Cl secretion due to the change in the electrochemical gradient induced by K+ efflux.

Similar to the controversy surrounding CaCCs, ClC-2-mediated Cl secretion has been measured in intestinal epithelial cell lines and mouse intestine; however, ClC-2 KO mice do not exhibit intestinal obstruction, and the ClC-2/CFTR double KO mice have the same intestinal phenotype as CFTR KO mice (Zdebik et al., 2004). Furthermore, a recent study did not observe altered cAMP and Ca2+-mediated Cl secretion in ClC-2 KO mice but rather that ClC-2 was required for electroneutral NaCl and KCl absorption in the colon (Catalan et al., 2012). In addition, ClC-2 localization differs among models tested as well as between gut segments. For example, in the human intestine, ClC-2 localized to a juxtanuclear compartment in the small intestine and basolateral membrane in the colon (Lipecka et al., 2002). However, ClC-2 activity may be coupled with CFTR since lubiprostone stimulation of ClC-2 and Cl secretion required WT CFTR (Bijvelds et al., 2009).

2.2.5. The Enteric Nervous System


The enteric nervous system (ENS) is a complex network of intrinsic primary afferent neurons, interneurons, and motor neurons that regulate the movement of water and electrolytes between the intestinal lumen and the bloodstream. Intrinsic primary afferent neurons detect physical changes such as mechanical stress/tension in the intestinal mucosa as well as luminal chemical changes that occur during normal digestion (Furness et al., 1999). In response to these changes, intrinsic primary afferent neurons generate reflexes to control motility, secretion and blood flow. Secretomotor (type of motor) neurons innervate the small intestinal and colonic crypts and goblet cells to control ion permeability to balance fluid secretion with absorptive processes (Furness et al., 1999). The release of neurotransmitters acetylcholine and vasoactive intestinal peptide by secretomotor neurons occurs in response to sympathetic innervations to stimulate Cl and HCO3 secretion (Reddix et al., 1994). Stimulation of epithelial anion secretion by secretomotor neurons is linked with vasodilation, which serves as the source of some of the secreted fluid.

Several studies have demonstrated that activation of the ENS stimulates intestinal fluid secretion during diarrheal diseases. In fact, 50% of fluid loss in secretory diarrheas, including rotavirus, is neuronally mediated (Lundgren, 2002Lundgren and Jodal, 1997Lundgren et al., 2000). In response to enteric pathogens, enteroendocrine cells release peptides or hormones (eg, serotonin, secretin, guanylin, etc.) which act on secretomotor neurons to induce fluid loss and increased motility (Furness et al., 1999). Overstimulation of secretion in response to enteric pathogens may lead to dehydration and possibly death.

3. Gastrointestinal pathophysiology


Diarrhea is attributed to one or more of the following mechanisms that drive the excessive loss of fluid: osmosis, active secretion, exudation, and abnormal motility (Field, 2003). Osmotic diarrhea may occur when high concentrations of poorly absorbable solutes, such as lactulose, sorbitol, or magnesium, generate an osmotic gradient, driving mucosal to luminal fluid flow. Mutations in nutrient transport or metabolism (eg, lactose deficiency) that prevent digestion of otherwise absorbable nutrients (typically glucose or galactose) can also result in osmotic diarrhea. In the colon, nonabsorbable carbohydrates are metabolized by commensal microbes into short chain fatty acids such as propionate, butyrate, and acetate in concentrations higher than the fluid absorptive capacity of the colon. Secretory diarrhea is characterized by large stool volumes (>1 L) that lack red and white blood cells and an osmotic gap in electrolytes. Na+, K+, Cl, and HCO3 are the major luminal solutes which are either malabsorbed or hypersecreted in response to pathologic secretory stimuli including bacterial toxins, inflammatory mediators, or endocrine tumors (eg, carcinoid syndrome, VIPoma) (Montrose et al., 1999). Exudative diarrhea refers to the disruption of intestinal epithelial barrier integrity that occurs due to either the excess loss of epithelial cells or dysregulation of tight junction function. Under these conditions, increased hydrostatic pressure in blood vessels and lymphatics leak water and electrolytes as well as lose protein and blood cells to the intestinal lumen (Field, 2003). In motility disorders, increased and decreased motor functions have been found to induce diarrhea. Decreased motility can result in bacterial overgrowth that compromises the normal digestive capacity of the small intestine. Increased bacterial colonization results in deconjugation and rapid reabsorption of bile acids which effectively decreases the concentration of bile salts required for fat absorption (Mathias and Clench, 1985). Moreover, bacteria can also induce carbohydrate malabsorption by inactivating BB oligosacchridases necessary for epithelial uptake (Mathias and Clench, 1985).

Comparison of the four major viral causes of gastroenteritis (ie, rotaviruses, noroviruses, astroviruses, and enteric adenoviruses) in humans reveals similar pathologic characteristics such as site of infection, mechanisms of action, and type of diarrhea. These enteric viruses invade the intestinal epithelium of the small intestine, elevate intracellular Ca2+ concentrations, decrease nutrient absorption, stimulate Cl secretion, and induce epithelial cell loss, which all contribute to the moderate/severe diarrhea phenotype observed in human disease (Karst et al., 2015Moser and Schultz-Cherry, 2005Ramig, 2004). Each type of virus infection is associated with various extraintestinal phenotypes (eg, vomiting, nausea, viremia), and some of these are reviewed in Chapters 2.62.9, and 3.4. The ontogeny of viral gastroenteritis in humans has been difficult to characterize due to limited availability of patient tissue samples during the early stages of infection. Thus, much of our understanding of viral pathogenesis has come from animal models, cancer derived intestinal epithelial cell lines, and the infection of the African green monkey kidney cell line, MA104, and other cell lines. The use of multiple model systems has confounded some observations which identified altered cellular and/or molecular events as mechanisms for diarrhea (Lorrot and Vasseur, 2007). For example, some data have indicated that viral infections induce intestinal epithelial cell loss resulting in epithelial lesions and thus a decreased capacity to absorb water, ions, and nutrients due to decreased intestinal surface area (Davidson et al., 1977). However, more recent animal studies of rotavirus, norovirus and astrovirus diarrhea have suggested that diarrhea occurs in the presence of an intact intestinal epithelial barrier and prior to any evidence of epithelial loss (Karst et al., 2015Moser and Schultz-Cherry, 2005Ramig, 2004Lundgren and Svensson, 2001). Furthermore, decreased activity and amount of BB digestive enzymes were also observed in the absence of cell death (Jourdan et al., 1998). Taken together, these data suggest that viral pathogenesis disrupts normal intestinal epithelial physiology that may lead to the subsequent loss of epithelial cells observed in the later stages of viral infection. For further details see Chapter 2.4.

The lack of consistent evidence that epithelial damage is responsible for malabsorption and water loss suggests that viral-induced diarrhea may also result from alterations in intestinal absorption and secretion. Transport physiology studies designed to elucidate the exchangers, symporters, and/or channels responsible for the decreased ion and nutrient absorption and increase Cl secretion associated with viral gastroenteritis are incomplete. In rotavirus diarrhea, nutrient malabsorption is correlated with decreased expression of SGLT1 while increased Cl secretion has been suggested to involve CaCCs since RV-mediated Cl secretion can still occur in the absence of functional CFTR (Halaihel et al., 2000). A recent study reported that rotavirus can inhibit NHE3 activity as early as 30 min post infection in intestinal epithelial cells, suggesting that inhibition of electroneutral Na+ absorption may contribute to RV diarrhea (Foulke-Abel et al., 2014). Astrovirus, enteric adenovirus, and norovirus diarrhea also present with primary malabsorptive and secretory phenotypes (Karst et al., 2015Moser and Schultz-Cherry, 2005) and thus, additional transport studies are needed to identify the transporters involved.

Over the last two decades, considerable progress has been made to understand the molecular mechanisms responsible for normal ion and water absorption as well as for each of the previously described contributors to diarrhea. The identification and characterization of bacterial and viral enterotoxins (NSP4 for rotavirus; see Chapter 2.4) have provided valuable insights into the cellular and molecular mechanisms of diarrhea diseases. Mechanisms invoked include pathogen entry/invasion, further characterization of signal transduction pathways, identification of multiprotein regulatory complexes, predicting structure/function relationships, and protein trafficking (Hodges and Gill, 2010Viswanathan et al., 2009). For example, several intestinal ion transporters and channels, including NHE3 and CFTR, contain PDZ [post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 (ZO-1) protein] protein–protein interacting domains at their carboxy-terminal ends. These PDZ domains directly bind regulatory proteins that increase/decrease transporter/channel activity based on the binding partner. The NHERF (Na+/H+ exchanger regulatory factor) family of multi-PDZ domain containing proteins has been shown to regulate the activity of NHE3, CFTR, and DRA (among nearly 70 other targets) by affecting their turnover number and/or trafficking to and from the enterocyte BB (Donowitz et al., 2005). Other PDZ proteins have been shown to regulate tight junction function by affecting tight and adherence junctional complex formation, thereby modulating ion and small solute permeability through the paracellular network (Van Itallie and Anderson, 2014). Studies of bacterial infection, including enterotoxigenic, enteroaggregative, enterohemorrhagic, and enteropathogenic E. coli, have demonstrated decreased tight junction function due to mislocalization or extracellular damage of tight junction proteins (Nassour and Dubreuil, 2014Simonovic et al., 2000Strauman et al., 2010Tomson et al., 2004). The effects of enteric viruses on tight junction function are limited as these studies are difficult to assess in vivo. Studies in MDCK and Caco-2 cell lines have reported decreased TER and increase permeability to large cargo (20 kDa) after RV infection (Dickman et al., 2000Tafazoli et al., 2001). Whether these effects occur in other enteric viral infections in the human intestine remains to be determined.

Since the use of multiple models to study viral pathogens has limited our understanding of human disease, the recent development of the methodologies to culture primary human differentiated epithelial cells from the recently identified Lgr5+ stem cell has reinvigorated the study of gastrointestinal physiology and pathophysiology in general (Jung et al., 2011; Sato et al., 2011b). Primary human intestinal epithelial cultures may be derived from either human crypt based stem cells (termed enteroids/colonoids) or from a single inducible pluripotent stem cell (iPSC) line (termed intestinal organoid) (Jung et al., 2011; Sato et al., 2011b; McCracken et al., 2011). These cultures are comprised of the complete complement of intestinal epithelial cells including immature enterocytes (that may be differentiated into more mature absorptive enterocytes), enteroendocrine, goblet, Paneth, tuft, and Lgr5+ stem cells. Enteroid/colonoid cultures represent the intestinal segment from which they are derived, and recent modifications generating intestinal organoid cultures have developed organoids with proximal and distal intestinal phenotypes (Watson et al., 2014). Furthermore, the human enteroid model has been shown to perform Na+ absorptive and Cl secretory functions and is being used to understand the pathogenesis of host–pathogen interactions (Foulke-Abel et al., 2014Kovbasnjuk et al., 2013). Recent work by the laboratory of Mary Estes has demonstrated that intestinal organoids and enteroids may be infected by RV and that each model has the capacity to facilitate viral replication resulting in the production of infectious viral progeny (Finkbeiner et al., 2012Saxena et al., 2015). The development of enteroids/colonoids and intestinal organoids as models to study the human intestine offer the opportunity to study the pathogenesis of enteric viral infections.

4. Conclusions


Nearly 50 years of basic and clinical research has advanced our understanding of the complex regulation of fluid absorption that occurs under normal digestive conditions and in diarrheal diseases. However, much of this work has come from studies in animal models and transformed cell lines, and yet the most effective therapy for acute moderate to severe diarrheal diseases remains Oral Rehydration Solution (ORS). While ORS may save patients from life-threatening dehydration, it cannot prevent the long-term effects (eg, stunted growth, delayed intellectual development) that result from episodes of severe diarrhea (especially in children less than 5 years old). Therefore, additional medical therapies are needed to treat severe diarrheal diseases. Currently, our understanding of diarrhea due to enteric pathogens comes from studies of bacteria, and yet the mechanisms responsible for these secretory and enterotoxigenic diarrheas are not completely recapitulated in viral gastroenteritis, based on patient stool analysis and in vitro studies. In comparison to rotavirus diarrhea, similar patterns of pathogenesis (eg, site of infection, primary malabsorption, lack of histologic damage) have emerged relating to diarrhea phenotype, suggesting that similar mechanisms may be common among other types of enteric viruses. However, additional mechanistic studies are required to identify and characterize the membrane transport processes altered in each type of viral infection. The development of normal human intestinal epithelial models that are capable of viral replication now offers an opportunity to advance the field of viral gastroenteritis by providing a more relevant understanding of how enteric viruses affect normal human intestinal physiology and by hopefully uncovering novel therapeutic targets for future drug development.

5. Abbreviations


APN Aminopeptidase N


AQP Aquaporin


ASCT1 Na+-dependent neutral amino acid transporter


B0AT1 Na+-dependent neutral amino acid transporter


BB Brush border


BPO Benzopyrimido-pyrrolo-oxazine-dione


CaCC Calcium-activated chloride channel


CF Cystic fibrosis


CFTR Cystic fibrosis transmembrane regulator


cGMP Cyclic guanosine monophosphate


ClC2 Chloride channel 2


CTx Cholera toxin


Dlg1 Drosophila disc large tumor suppressor


DPPIV Dipeptidyl peptidase IV


DRA Down regulated in adenoma


ENaC Epithelial Na+ channel


ENS Enteric nervous system


GLUT2 Glucose transporter 2


GLUT5 Glucose transporter 5


GlyH Glycin hydrazide


iPSC Inducible pluripotent stem cell


KO Knock-out


Lgr5 Leucine-rich G-protein coupled receptor 5


MUC Mucin


nAA Neutral amino acid


NDO Nondigestible oligosaccharides


NHE3 Na+/H+ exchanger 3


NHERF Na+/H+ exchanger regulatory factor


Na + /K+ ATPase Na+/K+ adenosine triphosphatase


NKCC1 Na+/K+/2Cl symporter


ORS Oral rehydration solution


PAT1 Putative anion transporter 1


PEPT1 Peptide transporter 1


PDZ domain Postsynaptic density protein 95 (PSD-95)/Drosophila disc large tumor suppressor (Dlg1)/ZO-1


PPQ Pyrimido-pyrrolo-quinoxalinedione


RV rotavirus


SCFA Short chain fatty acids


SGLT-1 Na+-dependent glucose cotransporter


STa Heat stable enterotoxin


TER Transepithelial electrical resistance


TJ Tight junction


TMEM16A Anoctamin 1, calcium-activated chloride channel


ZO-1 Zona occludens 1



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Apr 25, 2018 | Posted by in MICROBIOLOGY | Comments Off on Gastrointestinal Physiology and Pathophysiology
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