Organisation of the intestinal mucosa

The intestinal luminal surface represents the largest area of the body that is in direct contact with environmental contaminants and microbial agents, and its most striking anatomical features are its segmental division and the organisation of the intestinal mucosa into the crypt-villus axis. The length of villi decreases from the proximal to the distal end of the intestine. For instance, in broiler chicken, the average villous length is 1.80 μm in the duodenum (Leeson et al., Reference LEESON, NAMKUNG, ANTONGIOVANNI and LEE2005; Chou et al., Reference CHOU, CHUNG and YU2009; Panda et al., Reference PANDA, RAMA RAO, RAJU and SHYAM SUNDER2009) and 1.08 μm in the colorectal region (Nasrin et al., Reference NASRIN, SIDDIQI, MASUM and WARES2012). The presence of a crypt-villus axis along the length of intestine has immediate functional implications. While the immature crypt epithelium is primarily involved in ion secretion, the mature villus epithelium derived from the crypts is involved in nutrient absorption. At the cellular level the epithelial lining is composed of a single layer of columnar epithelial cells and interepithelial tight junctions (TJ) present at the apical-most region of the paracellular space between adjacent enterocytes (Figure 1). Tight junctions consist of an array of membrane-spanning proteins (e.g., barrier forming occludin and pore forming claudins) that are linked to the cytoskeleton by the cytoplasmic plaque protein zona occludin. Thus, such an intestinal barrier, consisting of a surface epithelium with intercellular TJs, not only permits the selective movement of ions, nutrients and water, but also restricts the translocation of microbes and toxins from the lumen (Podolsky, Reference PODOLSKY1997).

Figure 1

Basic ion absorption mechanisms in intestinal epithelial cells. An efficient absorption of Na⁺ involves transcellular (multiple ion transporters on the apical and basolateral membrane) and paracellular routes.

Basic scheme of intestinal ion transport

The lipid bilayers in the plasma membrane of the intestinal epithelial cells are relatively impermeable to ions which necessitates the presence of special carriers on the cell membrane to facilitate the transport of ions from the intestinal lumen into the epithelial cells and vice versa. These special carrier proteins are predominantly either transporters or ion channels. Transporters are proteins which bind a specific solute and is subsequently moved across the membrane due to a conformational change in the protein while ion channels are proteins that form charge- and size-specific pores in the plasma membrane. This basic scheme of ion transport across the intestinal lining permits efficient ion homeostasis. Crucial to this process is the polarity of the epithelial cells conferred by the presence of the apical TJs which ensures that the apical and basolateral ion transporters remain in their respective locations in the cell membrane (Figure 1).

Ion transport at the membranes occurs as follows. At the basolateral membrane, an ion gradient generated by the ATP driven movement of Na⁺ and K⁺ via the Na⁺K⁺ATPase (sodium potassium ATPase, commonly referred to as the ‘sodium pump’) helps passive diffusion of Na⁺, K⁺, and Cl^– through the Na⁺-K⁺-Cl^- cotransporter NKCC1 (Figure 1). At the apical membrane, three major mechanisms of sodium absorption are present. The first mechanism involves the sodium glucose linked transporter (SGLT-1), which is stimulated by the presence of glucose in the lumen. Oral rehydration solutions containing sugar and salt make use of this mechanism to significantly increase the absorption of sodium. Importantly, this movement of sodium and glucose also establishes a gradient that stimulates the movement of sodium and water through the paracellular space, which is referred to as the ‘sodium drag’. The second mechanism pertains to the sodium hydrogen exchanger isoform-3 (NHE3) which is present throughout small intestine and exchanges one sodium ion for every proton. This is an important mechanism of post-prandial sodium absorption. The electrogenic sodium channels (ENaC) represent the third mechanism of sodium absorption and are important in the large intestine. ENaC are important targets of the renin-angiotensin-aldosterone system that regulates blood pressure and fluid balance.

Apart from these mechanisms, a cell electric potential that is 30-40 mV negative relative to the lumen provides an additional driving force for the coupled movement of Na⁺ with solutes such as amino acids, oligopeptides and bile acids through a separate set of transporters (Field, Reference FIELD2003). In the avian caecum, naturally occurring volatile fatty acids such as acetate and butyrate act as substrates for energy dependent sodium transport (Grubb, Reference GRUBB1991a), indicating efficient and segment-specific ion transport. The lower intestine (colon and coprodeum) of the domestic fowl represents a special intestinal segment that can be considered as serving a reserve function. Under demanding conditions, such as a low salt diet, the colon and the coprodeum undergo both morphological changes, such as an increase in surface area, and functional changes, such as the greater expression of the electrogenic sodium channels (Laverty et al., Reference LAVERTY, BJARNADOTTIR, ELBROND and ARNASON2001; Elbrond et al., Reference ELBROND, JONES and SKADHAUGE2004; Laverty et al., Reference LAVERTY, ELBROND, ARNASON and SKADHAUGE2006).

From a secretion point of view, the cystic fibrosis transmembrane conductance regulator (CFTR) is the major chloride channel present on the apical membrane (Figure 2). Genetic mutations in the CFTR are responsible for the notoriously serious human condition, Cystic Fibrosis. The K⁺ extruding potassium channels on the basolateral membrane (IK1, calcium activated potassium channel; KCN3, voltage gated potassium channel) helps maintain an intracellular electronegative potential which also aids in the diffusion of chloride through the apical channels. Besides CFTR, several other chloride channels, such as the Ca⁺ activated chloride channel (CLCA), the outwardly rectifying chloride channel (ORCC) and the chloride channel ClC-2 are present on the apical membrane. However, these channels may have a relatively minor role in chloride secretion compared to the CFTR. In chicken colonocytes, the anion exchanger (Cl/HCO3^-) and the Na⁺/HCO₃⁻ cotransporter, in association with the NHE, maintain ion homeostasis by efficiently exchanging Na⁺, Cl^–, H⁺, and HCO₃^– (Calonge and Ilundain, Reference CALONGE and ILUNDAIN1998).

Figure 2

Basic ion secretion mechanisms in intestinal epithelial cells. The Cl^– secretion is maintained through co-ordination between K⁺ channels and NKCC1 on basolateral side and CFTR on apical side.

It is important to mention here that several of these ion transport mechanisms are not well characterised in the avian intestine. For example, though the expression and activity of the CFTR Cl^– channel has been demonstrated in the renal proximal tubule of chickens (Laverty et al., Reference LAVERTY, ANTTILA, CARTY, REDDY, YUM and ARNASON2012), its identity in the chicken intestine has not yet been clearly established. Besides cellular and molecular biology techniques, many of the studies on intestinal ion transport are performed using the Ussing chambers, which mimic the intestinal in vivo transport function but in an ex vivo experimental setting. This technique utilises measurement of the short-circuit current as an indicator of net ion transport across an epithelium such as gut mucosa and measurement of the flux of paracellular inert probes such as mannitol or inulin as an indicator of epithelial paracellular permeability (Grubb, Reference GRUBB1991b; Clarke, Reference CLARKE2009).

Intestinal ion transport during development

In chickens, enterocyte proliferation is not strictly restricted to the crypts at hatching and the crypt-villus axis develops in the first five days after hatching (Uni et al., Reference UNI, GEYRA, BEN-HUR and SKLAN2000). The villous transit time (time required for the migration of cells from the crypt to the tip of the villus) was found to be 72 hours after two days of hatching but 96 hours after two of weeks of age (Uni et al., Reference UNI, GEYRA, BEN-HUR and SKLAN2000), indicating a rapid compartmentalisation of the intestinal mucosa immediately after the hatching. The Paneth cells residing in intestinal crypts of many mammals have an important role in cell renewal and immunity. Though existence of Paneth cells in the chicken intestine was not clear previously (Nile et al., Reference NILE, TOWNES, MICHAILIDIS, HIRST and HALL2004), a recent study based on ultrastructural examination and in-situ hybridisation for lysozyme c mRNA, demonstrated the presence of Paneth cells in chicken intestinal crypts (Wang et al., Reference WANG, LI, LI, LI, LV, LI, MI and ZHANG2016). During late incubation and around hatching, the intestinal mucosa strategically prepares for digesting carbohydrate- and protein-rich exogenous food, and it has been shown that the mRNA expression and activity of SGLT-1 and Na⁺K⁺ATPase significantly increase during this period (Uni et al., Reference UNI, TAKO, GAL-GARBER and SKLAN2003). Hitherto sterile, the chicken intestinal tract undergoes microbial colonisation immediately after hatching and the quality of such microbial colonisation can have an enormous impact on intestinal health and growth. Besides affecting growth and intestinal microbial composition (Nakphaichit et al., Reference NAKPHAICHIT, THANOMWONGWATTANA, PHRAEPHAISARN, SAKAMOTO, KEAWSOMPONG, NAKAYAMA and NITISINPRASERT2011), colonisation pattern and initial exposure to varied bacterial communities can also affect ion transport related gene expression in the chicken gut (Yin et al., Reference YIN, LEI, ZHU, LI, WU, ZHANG, GAO, ZHU and WANG2010).

Effect of feed and feed additives on intestinal ion transport

Intestinal epithelial ion transport adapts remarkably to changes in the diet. Hens maintained on a low-NaCl diet for more than three-weeks have been shown to not only increase transepithelial sodium transport but also acquire a 100% increase in microvillus surface area with relatively greater numbers of open sodium channels in the cell membranes of the lower intestine (Sodring Elbrond et al., Reference SODRING ELBROND, DANTZER, MAYHEW and SKADHAUGE1991). A low sodium diet increases Na⁺/H⁺ exchange in the chicken ileum and colon due to an increase in the expression and activity of the NHE2 transporter isoform (Donowitz et al., Reference DONOWITZ, DE LA HORRA, CALONGE, WOOD, DYER, GRIBBLE, DE MEDINA, TSE, SHIRAZI-BEECHEY and ILUNDAIN1998). Contrarily, SGLT-1 expression decreases within two days of feeding a modified low sodium diet. It has been suggested that while activity of the apical NHE2 transporter in the ileum and the colon is regulated by aldosterone, activity of SGLT-1 is regulated by arginine vasotocin (De La Horra et al., Reference DE LA HORRA, CANO, PERAL, CALONGE and ILUNDAIN2001). Low dietary sodium is known to reduce the activity and sodium affinity of the Na⁺K⁺-ATPase located on the basolateral membrane (Gal-Garber et al., Reference GAL-GARBER, MABJEESH, SKLAN and UNI2003). Dietary protein composition has been shown to influence the mRNA abundance of PepT1 (a peptide transporter 1), amino acid (AA) transporters and aminopeptidase N in the small intestine of the chicken (Gilbert et al., Reference GILBERT, LI, EMMERSON, WEBB and WONG2010). A reduction in the phosphorus content of the diet given to chickens from hatching till four days of age has been shown to significantly induce the mRNA expression of the sodium-phosphate Na-Pi cotransporter in the small intestine (Yan et al., Reference YAN, ANGEL and ASHWELL2007). Further, while dietary addition of phytate has been shown to hamper carbohydrase activity and overall digestive competence, the inclusion of phytase in the diet reversed these phytate-induced effects and led to an increase in the expression of amylase, brush border enzymes sucrase and maltase, and Na⁺K⁺-ATPase (Liu et al., Reference LIU, RU, LI and COWIESON2008). The dietary supplementation of a probiotic Lactobacillus spp. can positively modify the architecture of the intestinal mucosal surface, and basal and glucose stimulated ion transport (Awad et al., Reference AWAD, GHAREEB and BOHM2010). Although numerous studies have demonstrated that an inclusion of dietary probiotics positively affects growth, intestinal morphology, immunity and resistance to intestinal pathogenic organisms (Patterson and Burkholder, Reference PATTERSON and BURKHOLDER2003; Ashraf et al., Reference ASHRAF, ZANEB, YOUSAF, IJAZ, SOHAIL, MUTI, USMAN, IJAZ and REHMAN2013; Bai et al., Reference BAI, WU, DING, LEI, BAI, ZHANG and CHIO2013; Cao et al., Reference CAO, ZENG, CHEN, ZHOU, ZHANG, XIAO and YANG2013; Lei et al., Reference LEI, LI, YU, RAJPUT and LI2013), the effects of probiotics on avian intestinal ion transport remain largely undefined and thus warrant further studies. This is particularly important as pre- and probiotics are being increasingly added to poultry feed for the maintenance of gut health.

Intestinal ion transport and microbial infections

Gastrointestinal infections are the outcome of the interplay between various pathogen virulence factors and host defence mechanisms that can result in altered gut transport or impaired barrier function, both of which can manifest clinical symptoms such as diarrhoea. Intestinal ion transport is critical for maintaining homeostasis of the luminal microbial population as studies have shown that the loss of ion transporters such as the CFTR and the NHEs can cause dysbiosis and intestinal inflammation (Norkina et al., Reference NORKINA, BURNETT and DE LISLE2004; Larmonier et al., Reference LARMONIER, LAUBITZ, HILL, SHEHAB, LIPINSKI, MIDURA-KIELA, MCFADDEN, RAMALINGAM, HASSAN, GOLEBIEWSKI, BESSELSEN, GHISHAN and KIELA2013). Microbial infections affect intestinal ion transport, resulting in either hypersecretion or malabsorption, and utilize multiple pathways for the same. Many microbial agents are known to directly interact with individual intestinal ion transporters to either increase or decrease their activity and affect their presence on the apical membrane of the epithelial cells. Pathogenic microbial agents interact with TJs and the surrounding actin cytoskeleton to diminish intestinal barrier function. The loss of intestinal barrier function allows luminal antigens an increased access to the immune cells in lamina propria, which further activate the inflammatory cascade and exacerbate the diarrhoea. The inflammatory cytokines produced by the epithelial cells and the cells of the lamina propria are known to enhance intestinal secretion (Chang et al., Reference CHANG, MUSCH and MAYER1990). The effects of common microbial infections on avian intestinal ion transport are presented below.

Rotavirus

Rotavirus has been implicated as the causal organism of diarrhoea in chicken, turkey and several other avian species (Day et al., Reference DAY, SPACKMAN and PANTIN-JACKWOOD2007; Pantin-Jackwood et al., Reference PANTIN-JACKWOOD, SPACKMAN, DAY and RIVES2007). Infections can occur in any age group, can be subclinical or clinical, and cause morbidity and diarrhoea (McNulty et al., Reference MCNULTY, ALLAN and STUART1978). Recent studies attribute most of the effects of a rotavirus infection on intestinal ion transport to a viral enterotoxin which is a non-structural protein named NSP4. Though the diarrhoeagenic effects of the rotavirus NSP4 have not been directly studied in the avian intestine, a specific antigenic site (AS II, residues 136 to 150) has been shown to be conserved among a variety of mammalian and avian rotaviruses (Borgan et al., Reference BORGAN, MORI, ITO, SUGIYAMA and MINAMOTO2003) and the avian rotavirus NSP4 glycoproteins are known to be enterotoxic to suckling mice (Mori et al., Reference MORI, BORGAN, ITO, SUGIYAMA and MINAMOTO2002).

Astrovirus

Astroviruses cause morbidity and enteric infections in a number of mammalian hosts (Grohmann et al., Reference GROHMANN, GLASS, PEREIRA, MONROE, HIGHTOWER, WEBER and BRYAN1993; Glass et al., Reference GLASS, NOEL, MITCHELL, HERRMANN, BLACKLOW, PICKERING, DENNEHY, RUIZ-PALACIOS, DE GUERRERO and MONROE1996; Koci et al., Reference KOCI, MOSER, KELLEY, LARSEN, BROWN and SCHULTZ-CHERRY2003) and are known to be widely prevalent in commercial chicken and turkey flocks (Reynolds and Saif, Reference REYNOLDS and SAIF1986; Reynolds et al., Reference REYNOLDS, SAIF and THEIL1987; Baxendale and Mebatsion, Reference BAXENDALE and MEBATSION2004). Diarrhoea, retarded growth and altered intestinal maltase activity with minimal histologic changes have been described in the intestine of astrovirus-infected juvenile turkeys (Thouvenelle et al., Reference THOUVENELLE, HAYNES, SELL and REYNOLDS1995). The Turkey astrovirus, TAstV-2, originally isolated from natural outbreaks of complex diarrheal diseases involving multiple organisms, has been reported to cause experimental diarrhoea even in the absence of overt histological changes and enterocyte death (Koci et al., Reference KOCI, MOSER, KELLEY, LARSEN, BROWN and SCHULTZ-CHERRY2003). In our studies, TAstV-2 infection reduced the transport of Na⁺ by redistribution of the apical NHE3 (Na⁺-H⁺ exchanger) from the membrane to the cytosol which was, in turn, associated with a rearrangement of the F-actin filaments (Nighot et al., Reference NIGHOT, MOESER, ALI, BLIKSLAGER and KOCI2010). As opposed to secretory diarrhoea due to excessive Cl^– efflux, impaired Na⁺ absorption causes malabsorptive diarrhoea as the retention of Na⁺ in the lumen results in a reduction in the ion gradient required for efficient water absorption. The role of autocrine and paracrine cytokines is also important for maintaining intestinal homeostasis. For example, an increase in TGF-β activity, which preserves epithelial barrier function and dampens the cAMP-mediated Cl^– secretory response (Howe et al., Reference HOWE, GAULDIE and MCKAY2002), has been reported to occur within 24 hours of TAstV-2 infection and it remains elevated till 12 days post infection in turkey poults (Koci et al., Reference KOCI, MOSER, KELLEY, LARSEN, BROWN and SCHULTZ-CHERRY2003).

Other microbial infections

Asymptomatic infections can affect intestinal ion transport and compromise production efficiency in poultry. One such example is infection with Campylobacter jejuni, which has been shown to reduce the intestinal absorptive surface area, alter ion transport and reduce intestinal epithelial barrier function in chickens (Awad et al., Reference AWAD, MOLNAR, ASCHENBACH, GHAREEB, KHAYAL, HESS, LIEBHART, DUBLECZ and HESS2015). The effects of protozoan infections on intestinal ion transport in poultry is largely unknown but studies in other species and laboratory animals indicate that protozoan infections can profoundly affect intestinal ion transport. For instance, chronic cryptosporidiosis in immunocompromised mice has been shown to decrease net ileal ion transport, weaken the paracellular barrier and impair Na⁺-glucose cotransport (Kapel et al., Reference KAPEL, HUNEAU, MAGNE, TOME and GOBERT1997). During experimental infections with Eimeria separata in the rat, sodium absorption in the infected region of the proximal colon was impaired but a compensatory increase in sodium absorption was observed in the non-infected distal colon through the aldosterone-regulated ENaC channel (Cirak et al., Reference CIRAK, KOWALIK, BURGER, ZAHNER and CLAUSS2004). Severe infection with Spironucleus spp. in farmed pheasants and partridges has been shown to reduce intestinal ion absorption by more than half when compared to healthy birds (Lloyd et al., Reference LLOYD, IRVINE, EVES and GIBSON2005). Among mycotoxins, an acute exposure to aflatoxin AFB₁ has been shown to increase apical anion secretion in the jejunal epithelium of chickens with minimal effects on active glucose uptake (Yunus et al., Reference YUNUS, AWAD, KROGER, ZENTEK and BOHM2010). Various trichothecenes such as DON, NIV and 15-Ac-DON have been shown to be capable of reducing SGLT activity (Awad et al., Reference AWAD, BOHM, RAZZAZI-FAZELI, HULAN and ZENTEK2004; Reference AWAD, REHMAN, BOHM, RAZZAZI-FAZELI and ZENTEK2005; Reference AWAD, ASCHENBACH, SETYABUDI, RAZZAZI-FAZELI, BOHM and ZENTEK2007; Reference AWAD, RAZZAZI-FAZELI, BOHM and ZENTEK2008).

Adaptation of avian intestinal ion transport to nutritional and environmental alterations

There is substantial evidence that ion transport in the avian intestine adapts to nutritional or environmental changes. For example, dehydration results in an increase in sodium-hydrogen exchange, albeit with segmental differences; NHE2 activity increases in the ileum and the colon, while in the jejunum, the activity of both NHE2 and NHE3 increases. Dehydration also leads to an increase in serum aldosterone levels and sodium glucose co-transport in all intestinal segments (De La Horra et al., Reference DE LA HORRA, CALONGE and ILUNDAIN1998). Another example of intestinal adaptation is an increase in the intestinal uptake of Ca²⁺ and calcium binding protein after restricted feeding during moulting in hens (Berry and Brake, Reference BERRY and BRAKE1991; Al-Batshan et al., Reference AL-BATSHAN, SCHEIDELER, BLACK, GARLICH and ANDERSON1994). Pharmacologic stress, due to the administration of corticosterone, has been shown to result in a compensatory increase in intestinal absorptive function, i.e., an increase in the relative abundance of SGLT-1, vitamin D-dependent calcium-binding protein-28,000 molecular weight (CaBP-D28k), peptide transporter 1 (PepT-1) and the liver fatty acid-binding protein (L-FABP) (Hu et al., Reference HU, GUO, HUANG, BUN, ZHANG, LI, LIU, LONG, YANG and JIAO2010).

Adaptation of fast growing broilers to the exposure of ambient heat is an important matter for commercial poultry production. Broiler chicken exposed to warm temperatures (30°C) have been shown to exhibit an approximately 50% increase in the expression and activity of SGLT-1 in the jejunum. This is thought to be an adaptive response to heat because even though birds exposed to warm temperatures displayed reduced feed intake, pair-fed control birds (birds maintained at thermo-neutral temperature (20°C) but offered feed equal in amount to that consumed by warm temperature birds) did not exhibit such changes in the expression or activity of SGLT-1(Garriga et al., Reference GARRIGA, HUNTER, AMAT, PLANAS, MITCHELL and MORETO2006). Heat exposure reduced the gene expression of glucose transporter 2 (GLUT-2) which diffuses monosaccharides from the basolateral side of intestinal epithelial cells into the extracellular fluid, fatty acid binding protein FABP1, and fatty acid receptor CD36 in broiler chicken (Sun et al., Reference SUN, ZHANG, SHEIKHAHMADI, WANG, JIAO, LIN and SONG2015). The serum electrolyte and acid-base imbalances seen in broiler chicken exposed to heat are thought to be mediated by reduced activity of Na⁺K⁺ATPase, Mg₂⁺-activated ATPase, and Na⁺-ATPase in the intestine and kidney (Chen et al., Reference CHEN, SANGIAH, CHEN, RODER and SHEN1994). While ion transport and digestive activity is optimised to a certain degree in the face of reduced feed intake and decreased absorptive compartment (decreased intestinal surface area) caused by the heat exposure (Mitchell and Carlisle, Reference MITCHELL and CARLISLE1992), the threshold of physiological responses under such conditions requires further investigation.

Acknowledgements

The authors would like to thank Dr Fred Hoerr, Auburn, Alabama for invaluable inputs on this manuscript.