Pathophysiology of Intestinal Na⁺/H⁺ exchange

Abstract

Several members of the SLC9A family of Na⁺/H⁺ exchangers are expressed in the gut, with varying expression patterns and cellular localization. Not only do they participate in the regulation of basic epithelial cell functions, including control of transepithelial Na⁺ absorption, intracellular pH (pH_i), cell volume, and nutrient absorption, but also in cellular proliferation, migration, and apoptosis. Additionally, they modulate the extracellular milieu in order to facilitate other nutrient absorption and to regulate the intestinal microbial microenvironment.

Na⁺/H⁺ exchangers are frequent targets of inhibition in gastrointestinal pathologies, either by intrinsic factors (e.g. bile acids, inflammatory mediators) or infectious agents and associated microbial toxins. Based on emerging evidence, disruption of NHE activity via impaired expression or function of respective isoforms may contribute not only to local and systemic electrolyte imbalance, but also to the disease severity via multiple mechanisms. Here, we review the current state of knowledge about the roles Na⁺/H⁺ exchangers play in the pathogenesis of disorders of diverse origin and affecting a range of GI tissues.

Introduction

Na⁺/H⁺ exchange (NHE) is an evolutionarily conserved membrane transport mechanism attributed to members of the CPA (cation/proton antiporters) superfamily. ^1,2 Among the four inclusive families: CPA1, CPA2, PSE, and NaT-DC, the CPA1 (SLC9A) family contains the best-characterized plasmalemmal and intracellular isoforms (SLC9A1–9). Additionally, the SLC9B subgroup consists of two Na⁺/H⁺ antiporter (NHA) isoforms, NHA1 and NHA2 (SLC9B1 and SLC9B2). In the gut, expression of all NHE isoforms but NHE5 has been described. NHE1 (SLC9A1), NHE2 (SLC9A2), NHE3 (SLC9A3), and NHE8 (SLC9A8) have not only been implicated in regulating the basic functions of the epithelial cells, including control of the intracellular pH (pH_i), acidic mucosal microclimate, cell volume, and nutrient absorption, but also in cell proliferation³, cell migration^{3, 4}, and apoptosis.⁵ Intestinal expression of NHE4 (SLC9A4) in the small and large intestine remains controversial. While gastric expression and function has been confirmed,^{6, 7} its presence in the intestine, at least in rodents, was contested.⁷ In light of these observations, the interpretation of the more recent studies on the functional contribution of NHE4 to pH regulation in the human colonic crypts^{8, 9} has to be done with care, although species differences may account for the reported discrepancies.

As a principle, Na⁺/H⁺ exchangers utilize energy stored in the form of the electrochemical Na⁺ concentration gradient developed across the plasma membrane (secondary active transport) by the basolateral Na⁺/K⁺-ATPase (NKA). NHE is a mechanism critical for transepithelial movement of Na⁺ and HCO₃⁻ (and thus for luminal and systemic salt, volume, and acid-base homeostasis) (Figure 1). It also serves supporting roles for other nutrient transporters by providing the proton gradient for the proton-coupled absorption of amino-acids, peptides, organic anions, short-chain fatty acids, folate, and iron^{10, 11}. Only mutations in the NHE6 or NHE9 genes have been linked to human disease (X-linked mental retardation and familial autism, respectively).^{12, 13} Although long assumed, only recently Janecke et al.¹⁴ demonstrated novel recessive missense, splicing, and truncation mutations in the NHE3-encoding SLC9A3 gene as being linked to the pathogenesis of congenital sodium diarrhea. Non-genetically based functional alterations of intestinal NHE’s have also been linked with epithelial dysfunction. Details on molecular mechanisms of regulation of expression and function of NHEs are beyond the scope and limit of this article, but have been comprehensively covered in other reviews.^15–18 In this review, we summarize the current state of knowledge regarding the physiological roles of NHE isoforms in the gut, and roles that aberrant epithelial Na⁺/H⁺ exchange plays in the context of gastrointestinal disorder pathogenesis.

Figure 1. Simplified depiction of the apical Na⁺/H⁺ exchange in transepithelial sodium, glucose, and water absorption.

The inward Na⁺ gradient (low intracellular [Na⁺]) is maintained by the basolateral Na⁺/K⁺-ATPase (NKA), which provides the driving force for several secondary active transporters membrane transport proteins that import glucose, amino acids (not shown), and other nutrients into the cell by use of the sodium gradient. The intracellular concentration of potassium is coordinated by basolateral efflux via Kir7.1 (Potassium inwardly-rectifying channel, subfamily J, member 13, KCNJ13). Electroneutral NaCl absorption is driven by parallel Na⁺/H⁺ and Cl⁻/HCO₃⁻ exchange, the latter mediated by DRA (Down-regulated in adenoma, SLC26A3), and PAT1 (putative anion transporter 1, SLC26A6). This coupled activity is made possible by carbonic anhydrase, which provides intracellular HCO₃⁻. NHEs are cross-regulated with SLC26 family members and the Cl⁻ and HCO₃ transporting cystic fibrosis transmembrane conductance regulator (CFTR). In addition to Na⁺/D-glucose apical transport, SGLT1 activity stimulates NHE3 via an increased amount of apical NHE3 (see section on ORS). GLUT2 (Glucose transporter 2, SLC2A2) provide a basolateral exit route for glucose. ClC-2 (Chloride channel protein 2, CLCN2) is capable of basolateral Cl⁻ secretion, though its key role appears to be the regulation of intestinal barrier function by altering tight junction composition and recovery from injury. Additional route of electrogenic Na⁺ transport is provided by apical Na⁺ channels (ENaC; not shown)

1. Na⁺/H⁺ exchange in esophageal and gastric pathology

The role of NHE1 in the esophageal epithelium in Barrett’s esophagus and in the progression to esophageal adenocarcinoma

Barrett’s esophagus is a precancerous condition characterized by the metaplasia of columnar epithelium with goblet cells that replace the normal squamous stratified epithelium in response to chronic reflux of gastric acid and bile acids. Though it is thought to represent a protective adaptive response to noxious components of the gastric chyme, metaplasia is also associated with increased risk of esophageal adenocarcinoma. When esophageal luminal acidity reaches pH 2.0, the microenvironment adjacent to the surface cells of the stratified squamous epithelium is reduced to a pH of 2.0–3.0¹⁹. Upon exposure to acid, esophageal epithelial cells regulate their pH primarily via the combined actions of Na⁺/H⁺ antiport and Na⁺-dependent Cl⁻/HCO₃⁻ exchange²⁰. NHE1 is the sole plasmalemmal Na⁺/H⁺ exchanger isoform expressed in the rabbit and rat esophagus²¹ and is allosterically regulated (activated) by reduced pH_i in a PKC-dependent mechanism.²² Additionally, epidermal growth factor (EGF), abundant in the saliva, exerts a cytoprotective effect in acid-exposed cells via Ca²⁺/calmodulin- and PKC-dependent activation of NHE1²³. Consistent with that, patients with low salivary EGF levels were found to be susceptible to severe oesophageal damage as a potential consequence of gastroesophageal reflux and to form a high-risk group for development of Barrett’s oesophagus^{24, 25}. NHE1 expression is increased in patients with gastroesophageal reflux disease²⁶ and in Barrett’s esophagus²⁷, where it likely represents a cellular defensive mechanism to manage the acute and chronic acid overload. Bile acids present in reflux chyme reduce the ability of the cells to control their pH_i by nitric oxide-mediated NHE1 inhibition, thus leading to increased DNA damage and potentially to mutations and cancer progression.²⁷

However, NHE1 has diverse physiological roles extending well beyond pH_i and cell volume control, including cell proliferation, growth, migration, and apoptosis, and contributes to pathological processes such as cancer cell invasion and heart failure^{28, 29}. In a Barrett’s adenocarcinoma cell line acid pulse-induced NHE1 activity correlated with increased proliferation, which could be reduced by inhibition of NHE1 or PKC³⁰. This finding led to a somewhat paradoxical proposal that NHE1 inhibition may be of therapeutic value in Barrett’s esophagus and prevention of its progression to cancer.

NHE’s in gastric epithelial injury and repair

The integrity of the gastric epithelium is essential for protection from noxious luminal contents, which include gastric acid, proteases, and food-borne pathogens. As a consequence of damage to the surface epithelium, adjacent healthy cells rapidly migrate into the injured area to restore the mucosal barrier, and failure of this restitution process may lead to ulceration. Expression and activity of NHE1, and to a lesser extent NHE2 and NHE3, has been described in gastric myofibroblasts, cells that play a central role in wound healing, deposition of the extracellular matrix, and epithelial function.³¹ These functions are tightly correlated with the myofibroblasts’ ability to migrate and proliferate within the subepithelial compartment. NHE1 and its stimulation by insulin-like growth factor-II (IGF-II) and carbachol has been postulated to contribute to human gastric myofibroblast (HGF) migration and to be indispensable for IGF-II-induced HGF proliferation.³¹ Gastric epithelium expresses five NHE isoforms: apical NHE2 (in surface/neck mucous cells), apical NHE3 (in rat but not rabbit parietal cells), apical NHE8 (mouse fundic and pyloric glands), basolateral NHE1 (all cells), and basolateral NHE4 (in parietal and chief cells). ^32–34 Initially, NHE1 has been implicated in gastric repair in guinea pig mucosa in vitro.^{35, 36} Later studies with more definitive mouse knockout models, and the use of intravital two-photon laser microscopy following injury, showed that it is NHE2 and not NHE1 that is required for the cytoprotective effects of trefoil factor 3 in the gastric epitheliaum.³⁷ These findings were also in agreement with the in vitro studies by Furukawa et al.³⁸, and in the description of progressive gastritis and gastric atrophy with loss of parietal cells in NHE2^−/− mice.^{39, 40} Consistent with its expression pattern, loss of NHE4 in knockout mice resulted in reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells.⁶ More recent studies from our laboratory also showed that gastric expression of NHE8 was critical for the maintenance of bicarbonate secretion by the Cl⁻/HCO₃⁻ exchanger downregulated-in-adenoma (DRA; SLC26A3) and the associated mucosal protection. Compared to their wild-type littermates, mice lacking NHE8 had a higher incidence of gastric ulcer formation. ³² Although the mechanism responsible for DRA downregulation is not yet clear, it appears to be a phenomenon consistent across organs, as it was described also in the colon,⁴¹ conjunctiva, the cornea, and the lacrimal glands.⁴²

2. Intestinal Na⁺/H⁺ exchange in diarrheal diseases

Congenital sodium diarrhea

Congenital sodium diarrhea (CSD) is a rare autosomal recessive diarrheal disorder originally described in two patients in 1984 and 1985.^{43, 44} To date, there have been fewer than 50 CSD cases reported in the literature.⁴⁵ Prior to birth, polyhydroamnios (an excess of amniotic fluid in the amniotic sac) is observed, which is suggestive of an increase in colonic output by the fetus. After birth the disease is characterized by the secretion of a large volume of ‘nonstopping’ high Na⁺ diarrhea, often mistaken for urine, which leads to irritability, dehydration, and metabolic acidosis.⁴⁵ Although profuse diarrhea continues, electrolyte replacement therapy promotes normal growth and development.⁴⁶

Two of the early reports of CSD linked diarrhea to defective NHE in the jejunum^{44, 46}, although the identity of the transporter remained unknown. In an attempt to identify the specific isoform, Muller et al studied a small CSD cohort of five infants from two inbred Austrian families and found no contribution from any of the six then known NHE’s⁴⁷. Subsequent analyses demonstrated additional intestinal and corneal pathophysiology (intestinal epithelia dysplasia and corneal epithelial erosions) among other symptoms accompanying high Na⁺ diarrhea. These affect about a third of CSD patients, are referred to as the syndromic CSD form, and have been linked to a mutation in SPINT2.⁴⁸ The classical or non-syndromic form of CSD is strongly linked to defective NHE activity, and recently, using a cohort of 18 patients from 16 families, Janecke et al. determined that a variety of mutations (point, missense, and truncation) in the NHE3-encoding SLC9A3 gene occurred in a subset (9/18) of the studied CSD cases.⁴⁵ The identified SLC9A3 mutations included one whole-gene deletion, one splicing and two frame-shift mutations, all of which are expected to abolish protein production. Four missense mutations/variants, p.Arg382Gln, p.Ala311Val, p.Ala269Thr, p.Ala127Thr were tested in vitro, and all but p.Ala127Thr (benign variant) conferred decreased basal Na⁺/H⁺ exchange activity. ⁴⁵

Additionally, an activating mutation in the catalytic domain of the guanylate cyclase c gene (GUCY2C) was also identified and may account for 20% of sporadic CSD cases.^{49, 50} This mutation is likely mechanistically linked to hyperactivation of the cystic fibrosis transmembrane regulator (CFTR)⁴⁹ and reduced NHE3 function via elevated intracellular cGMP and NHE3 inhibition via a cGKII kinase-dependent mechanism.^{51, 52} Consequently, NHE3 inhibition may contribute to at least 70% of CSD cases. Additional research is required to identify the remaining contributing factors, especially in the fetal and neonatal period when the contribution of NHE3 to total epithelial NHE is thought to be minimal. Although NHE8 has a reciprocal ontogenic pattern of pattern of expression, with higher levels in young than older animals^{53, 54}, no mutations were found in the exonic regions of SLC9A8 gene in a small cohort of five CSD patients.⁵⁴

Inhibition of Na⁺/H⁺ exchange during infectious diarrhea

Diarrhea caused by enteric infections is a major factor in morbidity and mortality worldwide. The mechanisms are multifactorial and include alterations in motility, changes in paracellular permeability, loss of absorptive surface, and changes in electrolyte fluxes. A significant component of altered electrolyte flux in infectious diarrhea is increased chloride secretion via activation of apical chloride channels, including the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca²⁺-activated Cl⁻ channels.⁵⁵ However, there is a significant component or subset of infectious diarrheal cases related to inhibition of Na⁺ and fluid absorption. The underlying mechanism for the latter is a reduction in Na⁺ absorption via impaired NHE activity, with the affected NHE isoforms and the mechanisms involved varying among pathogens. During infection with Vibrio cholerae, a prime example of secretory diarrhea, cholera toxin is capable of reducing epithelial Na⁺ absorption via cAMP-dependent inhibition of NHE2 and NHE3.⁵⁶ Extrapolating from the known mechanisms governing NHE regulation^{57, 58}, one could generalize that bacterial pathogens which increase intracellular Ca²⁺, cAMP, and/or cGMP concentration inhibit electroneutral NaCl absorption. Indeed, this was shown in mouse models of infection with Salmonella typhimurium, Shigella dysenteriae type 1 toxin, and Campylobacter jejuni.^59–61 However, other mechanisms leading to NHE inhibition have also been described.

Clostridium difficile, the leading cause of nosocomial diarrhea and pseudomembranous colitis, also exerts inhibitory effects on epithelial NHE. C. difficile Toxin B (TxB) inhibits Rho-family GTPases and alters the interaction between NHE3 and the microvillar actin cytoskeleton to facilitate its internalization and loss of function.⁶² TxB was also shown to disrupt the stimulating effects of β-PIX (Rho Guanine Nucleotide Exchange Factor 7), which along with Shank2 maintains apical membrane NHE3 expression.⁶³ More recently, Engevik et al.⁶⁴ postulated that NHE3 inhibition by C. difficile toxin resulted in alteration of the intestinal environment and gut microbiota, which could facilitate colonization and expansion of the pathogen. They showed that when compared to healthy controls, decreased expression of NHE3 in infected patients correlated with elevated Na⁺ and pH of the stools, increased Bacteroidetes and Proteobacteria, and decreased Firmicutes phyla. Interestingly, in vitro growth of C. difficile was promoted by elevated Na⁺ and an alkaline media pH; consequently, the authors concluded that inhibition of NHE3 creates an altered environment favored by C. difficile.⁶⁴

Several classes of pathogenic E. coli strains are responsible for diarrheal outbreaks. These include enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli, and diffusely adherent E. coli (DAEC). A variety of NHE-independent mechanisms that contribute to diarrheagenic effects of EPEC are known (outside the scope of this review).⁶⁵ Surprisingly, the effects of EPEC in vitro (Caco-2 BBE, HT-29 cells and T84 cells) were isoform-specific, with significant increase in NHE1 (basolateral) and NHE2 (apical) activity and a concurrent 50% inhibition of NHE3.⁶⁶ In a follow-up study, Hodges et al.⁶⁷ showed that the inhibitory effects of EPEC on NHE3 were dependent on EspF, a component of EPEC type III secretion system. On the host side, Chen et al.⁶⁸ showed that E. coli heat-stable enterotoxin (STp), which binds to guanylate cyclase-C in the luminal enterocyte membrane and increases intracellular cGMP levels,⁶⁹ requires the presence of the regulatory PDZ adaptor protein NHERF2.⁶⁸ The downstream effector of elevated cGMP, type II isoform of cGMP-dependent protein kinase cGKII is a part of a NHE3 signaling complex,⁷⁰ and inhibition of NHE3 activity by cGMP/cGKII requires NHE3 protein phosphorylation at Ser⁵⁵⁴, and Ser⁶⁰⁷,⁵² although the relationship of NHERF2/ cGMP/cGKII in the context of exposure to E. coli enterotoxin is not yet clear. Increased apical NHE2 activity may be compensatory to the loss of NHE3, although it remains unknown whether it outweighs NHE3 inhibition in vivo. Contrary to NHE3^−/− mice⁷¹, targeting of SLC9A2 gene did not result in diarrhea^{39, 72}, suggesting that NHE2 is not a major absorptive Na⁺/H⁺ exchanger. Inhibition of NHE4 in T-84 cells by the heat-stable enterotoxin of enterotoxigenic E. coli has also been reported, although the relevance of this finding is unclear considering inconsistent the data on intestinal expression of this isoform. ^{7, 9}

Less is known about the role of NHE in diarrhea during viral gastroenteritis. Astroviral infections have been associated with Na⁺ malabsorption, and one study suggested that infection leads to decreased levels of NHE3 in the insoluble protein fraction in the enterocytes, presumed to represent the apical membrane.⁷³ Recently communicated data from rotavirus-infected patients showed that both NHE2 and NHE3 proteins are downregulated, and the remaining NHE3 is mislocalized.⁷⁴

Contribution of Na⁺/H⁺ exchange to the efficacy of oral rehydration therapy

The glucose transporter SGLT1 uses a Na⁺ gradient to transport Na⁺ and glucose at a 2:1 stoichiometric ratio against a glucose gradient.⁷⁵ In each cycle, a sugar molecule is co-transported with Na⁺ across the cell, which is accompanied by 260 water molecules.⁷⁶ This mechanism was calculated to account for 5 liters of water absorbed per day in the human intestine and formed the molecular basis of oral rehydration therapy aimed to control mortality associated with cholera and other infectious diarrheal diseases.⁷⁷ Lin et al.⁷⁸ demonstrated a key NHE3 contribution to the efficacy of oral rehydration solution. They showed that SGLT1-mediated Na-glucose co-transport stimulates NHE3 activity in vivo by an Akt/PKB- and NHERF2-dependent pathway. This increase was associated with increased brush-border NHE3 recruitment through its release from the endosomal storage pool. Activation of NHE3 by glucose reversed cholera toxin-induced NHE3 inhibition, a phenomenon that may at least partially explain the efficacy of oral rehydration solutions (ORS).⁷⁸ In 2004, both the World Health Organization and the United Nations Children’s Fund suggested the supplementation of ORS with zinc. This recommendation was based on observations linking a reduction in tissue and an increase in fecal zinc levels in infants and children with diarrhea⁷⁹ and that zinc deficiency is responsible for decreased net water and sodium transport from the small and large intestine.⁸⁰ Clinical studies showed a decline in diarrheic stool volume and episode duration when zinc was administered in conjunction with ORS.⁸¹ In addition to the anti-secretory effects of zinc⁸², Hoque et al. showed that zinc not only stimulated basal NHE3 activity by 50%, but it also counteracted forskolin-induced cAMP-mediated NHE3 inhibition.⁸³ While conventional ORS targets primarily small intestinal Na⁺ and water absorption, an improved formulation has been proposed to also target the colon.⁸⁴ In this approach, D-glucose is replaced with a relatively amylase resistant cornstarch. While some of the starch is enzymatically broken down in the jejunum to stimulate Na⁺ absorption, most of it would enter the colon where it could be fermented to short chain fatty acids (SCFA) such as propionate, butyrate and acetate by the resident bacteria. Butyrate increases expression and/or activity of apical NHE’s^85–88 and aids in transepithelial Na⁺ absorption via a neutral linked Na⁺ absorptive process that exchanges SCFA for OH⁻ ions along with the apical Na⁺/H⁺ exchange. In a small, randomized clinical trial, this ORS formulation performed better than standard ORS by reducing both the duration and volume loss of severe acute diarrhea.⁸⁴ Two other randomized controlled trials with the addition of high-amylose maize starch (HAMS) to ORS carried out in South India have demonstrated a substantial decrease in diarrhea duration in both adults and children hospitalized for acute diarrhea.^{89, 90} One drawback of such modified ORS is the precipitation of the starch in the ORS formulation. New formulations containing an anti-settling agent have been proposed and clinical trials have been reported in 2014 by Binder et al.⁹¹ as under way.

Modulation of intestinal Na⁺/H⁺ exchange in noninfectious diarrhea associated with immunosuppression (rapamycin)

Rapamycin or Sirolimus is a drug isolated from the actinomycete Streptomyces hygroscopius that exhibits immunosuppressive, antiproliferative, and antifungal properties. Rapamycin and its analogs (Rapalogs) have been shown to inhibit the activity of mTOR (mammalian or mechanistic Target of Rapamycin).⁹² mTOR is a serine-threonine kinase that is known to form two different catalytic complexes, the mTOR Complex 1 or 2 (mTORC1 or mTORC2) and to induce autophagy.⁹² Rapamycin administration inhibits mTORC1, while long-term use partially inhibits mTORC2. Its use to prevent organ transplant rejection and to treat lymphangioleiomyomatosis is associated with several side effects, which include diarrhea. Among patients receiving post-renal transplant rapamycin treatment, in those hospitalized for noninfectious diarrhea, ileal NHE3 expression was reduced at the apical membrane.⁹³ Of 367 records of renal transplant cases at the Albany Medical College, 20 patients had 39 events of acute, severe diarrhea requiring hospitalization. In all events, rapamycin levels at the time of diarrhea were significantly elevated (2–7 times higher than the baseline levels), and in all cases, diarrhea resolved within 3–5 days after the serum rapamycin levels returned to baseline.⁹³ This finding was reproduced in rapamycin-treated wild-type, but not in autophagy-resistant Atg7^−/− mice. Deletion of mTOR in mice also led to reduced NHE activity, luminal accumulation of fluid, and reduced levels of NHE3 and NHERF1.⁹³

3. Intestinal Na⁺/H⁺ exchange in clinical and experimental Inflammatory Bowel Diseases

Inflammatory Bowel Disease (IBD) is a group of chronic inflammatory diseases that include Crohn’s disease (CD) and ulcerative colitis (UC). Both conditions are characterized by immune system activation, microbial dysbiosis, and disruption of the epithelial barrier, which may lead to bacterial translocation, the latter being a more prominent feature of CD.⁹⁴ Alterations in the function of epithelial cells in IBD extend into transcellular nutrient transport.⁹⁵ In some instances, those alterations of transport protein complexes at the apical or basolateral membranes have consequences transcending their basal functions as nutrient or electrolyte transporters to modulate epithelial barrier function and gut microbiota, potentially shaping the immune response in the course of IBD.

Inhibition of intestinal Na⁺/H⁺ exchange as a contributor to inflammation-associated diarrhea

Diarrhea is one of the common symptoms in patients with IBD, occurring in about 50% of acute flare-ups in CD and in nearly all UC patients.⁹⁶ The pathophysiology of diarrhea in IBD is complex and multifactorial. It involves altered intestinal motility as well as abnormal epithelial ion transport and defective Na⁺ absorption in the colonic mucosa in particular.^{97, 98} Contrary to infectious diarrhea where active and excessive Cl⁻ secretion is predominant, in IBD, both electrogenic Na⁺ transport mediated by sodium channels^99–101 as well as electroneutral NHE and coupled Na-Cl absorption are reduced.¹⁰² . At least for UC, a model was proposed whereby the associated diarrhea is the result of a combination of factors: increased paracellular permeability to monovalent ions, reduction of electrogenic Na⁺ absorption (secondary to decreased apical Na⁺ channel and basolateral NKA expression), electroneutral Na⁺ absorption and coupled Na⁺-H⁺/ Cl⁻-HCO₃⁻ exchange, and as a result compromised active Cl⁻ absorption.¹⁰²

The mechanisms involved in the inhibition of epithelial NHE’s in IBD are not well defined and conflicting results have been published. IFN-γ inhibits the expression and function of NHE3 and NHE2 both in vitro and in vivo.¹⁰³ Inhibition of NHE3 expression and activity has also been described in several experimental models of colitis, including IL-2^−/− mice¹⁰⁴, and in DSS- and TNBS-induced colitis.¹⁰⁵ The latter study described decreased NHE3 protein expression in sigmoid mucosal biopsies from most of the cases of active UC and/or CD, in ileal mucosal biopsies of active CD, as well as in ~50% of sigmoid biopsies from inactive UC or CD. Siddique et al. showed that in both the untreated and treated patients with CD and UC, NHE3 protein and activity were also reduced.¹⁰⁶ Furthermore, NHE3 mRNA was reduced only in CD, but not in patients with UC. However, in IL-10^−/− mice NHE3 activity, measured in the apical enterocytes within isolated colonic crypts, was significantly decreased without altered NHE3 expression and localization.¹⁰⁷ The authors speculated that decreased expression of two key NHE3-regulatory PDZ adaptors NHERF2 and PDZK1 may be responsible for decreased NHE3 activity.¹⁰⁷ Additional studies with UC patients by Yeruva et al.¹⁰⁸ and Farkas et al.¹⁰⁹ showed a significant reduction of NHE3 activity despite the preserved protein and mRNA expression, thus arguing for post-translational regulation of the symporter activity. In an in vivo model of diarrhea mediated by anti-CD3 monoclonal antibody-induced T cell activation, Clayburgh et al.¹¹⁰ showed that in the jejunum, TNF induces NHE3 internalization. PKCα activation triggered by TNF was responsible for NHE3 internalization and the resulting Na⁺ malabsorption.¹¹⁰ While it is clear that the NHE3-mediated apical Na⁺/H⁺ exchange and epithelial Na⁺ absorption are inhibited in IBD, the exact mechanism remains unclear and may depend on the specific disease, segment involved, and/or the severity of inflammation.

NHE8 has a broader expression pattern than other apical NHE’s.^{53, 111, 112} Moreover, its expression is not limited to the apical membrane; in vitro studies with ectopically expressed NHE8 show it’s both present and functional in the trans-Golgi network and multivesicular bodies.¹¹³ It is broadly expressed in the colon, including the goblet cells, where it participates in the regulation of Muc2 expression and mucous secretion.^{41, 114, 115} Although we observed a dramatic decrease in the expression of NHE8 in UC patients (Li et al., unpublished data), loss of NHE8 expression and activity may not contribute to inflammation-associated diarrhea directly as NHE8^−/− mice do not exhibit symptoms of impaired Na⁺ absorption or diarrhea.⁴¹ The consequences of NHE8 deficiency in IBD may be more related to microbial-host interactions as discussed in the next section.

Both the role of the basolateral NHE1 and the potential changes in its expression and/or activity in the colonic epithelial cells in clinical and experimental IBD remains unclear, and the available data are inconsistent. Expression of NHE1 mRNA and protein was increased in both acetic acid and TNBS-induced colitis in rats,¹¹⁶ and non-specific pharmacological NHE inhibition with amiloride was deemed beneficial in rodent models of IBD.^{117, 118} However, in human patients, opposing data have been published. Contrary to the same group’s earlier rat data, Khan et al.¹¹⁹ and Siddique and Khan¹²⁰ showed decreased NHE1 mRNA in the biopsies of UC and CD patients, whereas Farkas et al.¹⁰⁹ showed consistently elevated NHE1 activity and expression in the inflamed mucosa of UC patients. The importance of these controversial findings remains unclear. While it may be consequential to some aspects of epithelial cell function, the notion that “NHE1 suppression may reduce an uptake of sodium chloride and water from the inflamed colonic lumen and thus contribute to diarrhea”¹¹⁹ currently lacks experimental support.

Apical Na⁺/H⁺ exchange as a modulator of the gut microbiome and inflammatory response during colitis

The lumen of the gastrointestinal tract is a habitat for 100 trillion bacterial cells, which form a virtual organ.¹²¹ This gastrointestinal bacterial consortium, collectively referred to as the gut microbiome, is a highly functional, complex, and dynamic entity. The composition of this virtual organ, known as the gut microbiota or microbiome, is generally conserved at the phylum level and is unique for each individual at lower taxonomic (genus and species) levels. The adult colon is predominantly colonized by two phyla Bacteroidetes and Firmicutes, followed by Proteobacteria and Actinobacteria.¹²² Cells of both the innate immune system (including epithelial cells) as well as of the adaptive immune system can sense microorganisms or their metabolic products and translate the signals into host physiological responses, which in turn can regulate the gut microbial ecology.^{123, 124} This cross-talk is essential for normal gut development, maturation of the immune system, optimal nutrient bioavailability, participation in xenobiotic and hormone metabolism, and the maintenance of the epithelial and immune cell microenvironment. Any perturbations in microbial-host homeostasis results in dysbiosis, a detrimental shift in the composition or microbial abundance, with consequences ranging from enteric infections^{125, 126}, colorectal cancer¹²⁷, and autoimmune diseases¹²⁸, through metabolic syndrome (type 2 diabetes and obesity)¹²⁹, to neuropsychiatric diseases and autism spectrum disorders.^130–133

Dysbiosis in both IBD patients and animal models is characterized by reduced general biodiversity,¹³⁴ expanded relative abundance of the Bacteroidetes, Proteobacteria and Actinobacteria phyla, decreased abundance of Firmicutes,¹³⁵ and importantly the presence of Clostridia spp. belonging to the clostridia IV and XIVa clusters, the major butyrate-producing bacteria and inducers of colonic CD4⁺FoxP3⁺ T regulatory cells.¹³⁶ Similar changes are observed in mouse models of IBD.^137–139 The mechanisms by which the gut microbial community is affected by the inflammatory process, or whether dysbiosis is sufficient to induce and/or modulate mucosal immune responses, remain a subject of debate. While some studies indicate that dysbiotic communities can be colitogenic upon transmission to wild-type mice, ¹⁴⁰ others have shown that mucosal bacteria from UC patients only confer susceptibility and are not sufficient to induce disease in untreated or otherwise non-genetically manipulated hosts.¹⁴¹

We hypothesized that inhibition of epithelial NHE, common in IBD patients, may lead to altered epithelial cell function and mucosal milieu that could lead or contribute to the observed shifts in microbial gut ecology and, as a consequence, influence mucosal inflammation. Indeed, mice lacking the dominant epithelial apical NHE isoform NHE3, when raised in a conventional facility, spontaneously develop distal colitis with mild diarrhea.¹⁴² Colitis symptoms were alleviated by broad-spectrum antibiotics and reduced by rederivation into a barrier facility.^{142, 143} NHE3^−/− mice develop microbial dysbiosis similar to IBD patients.¹⁴³ Although they appear to have normal mucous thickness, structure, and Muc2 mRNA expression, higher bacterial penetrance with increased bacterial adhesion to the mucosa and bacterial translocation was described, similar to IL-10^−/− mice.^{142, 144} These findings suggested that mucus thickness alone is not a major determinant of bacterial penetration, and that other factors may contribute to mucosal bacterial penetration in IBD.¹⁴⁴ It is plausible, however, that the molecular structure of MUC2 polymers within the inner layer may change with impaired apical Na⁺/H⁺ exchange. As MUC2 is packed into secretory granules in the goblet cells, the organellar pH changes from 7.2 in the ER to 6.0 in the trans-Golgi network and to 5.2 in the secretory granulae, along with increasing Ca²⁺ concentration.¹⁴⁵ Upon secretion, and before the 1,000-fold expansion, it remains densely packed to form the impenetrable for bacteria inner stratified layer, which is best preserved at pH 6.2 in form of sheets of N-terminal trimers.¹⁴⁵ Disruption of the regulatory mechanisms responsible for maintenance of the acidic microclimate could make the MUC2 network structure more penetrable for bacteria. This would, however, require experimental confirmation.

Importantly, NHE3^−/− mice are highly susceptible to experimental colitis, ^{146, 147} although the relative contributions of epithelial dysfunction and microbial dysbiosis are still unclear. Most recently, our group showed that in adoptive T cell transfer colitis NHE3 deficiency and the associated dysbiosis dramatically accelerated and exacerbated the disease, increased mucosal CD4⁺ T cell and neutrophil homing, and increased intestinal permeability, all of which was attenuated with broad-spectrum antibiotics.¹⁴⁸ Strikingly, a dysbiotic microbiome was more strongly associated with NHE3 deficiency than with T cell mediated colitis per se.¹⁴⁸ We concluded that Na⁺/H⁺ exchange is, therefore, a crucial process for maintaining microbial homeostasis within the gut and that its disruption in IBD is likely a strong contributor to dysbiosis, which in turn affects the progression and severity of disease. Another group also showed that higher ileal luminal Na⁺ concentration, and not changes in pH, is responsible for the relative expansion of Bacteroides thetaiotaomicron and expression and activity of fucosylase FUT2.¹⁴⁹ B. thetaiotaomicron has been shown to be colitigenic,¹⁵⁰ and an aberrantly high IL-8 response to B. thetaiotaomicron has been demonstrated in CD patients, ¹⁴⁹ findings that may provide a link between inhibition of NHE3 and mucosal neutrophil influx in IBD.

As suggested earlier in this review, NHE8 may also contribute to mucosal homeostasis through mechanisms distinct from that of NHE3, possibly through its yet undefined roles in the goblet cell function. Indeed, it is downregulated and confers higher susceptibility during experimental colitis with a Th2-like response.^{151, 152} Reduced expression or absence of NHE8 is also related to decreased Muc2 mRNA expression in goblets cells, and decreased mucosal expression of antimicrobial peptides.¹¹⁴ NHE8^−/− mice have reduced Muc2 expression, which could also be reproduced in colonic organoids from this strain (Xu et al. unpublished data), and show a reduced inner mucous layer and closer proximity of luminal bacteria to the brush border membrane.¹⁵² Consequently, loss of NHE8 in vivo led to higher adhesion of Salmonella typhimurium.¹⁵² In vitro, NHE8 siRNA knockdown showed similar increase in S. typhimurium adhesion, which appeared to be selective and did not affect adhesion of a probiotic strain of Lactobacillus plantarum JDM1.¹⁵² In summary, data from NHE3 and NHE8 knockout mice suggests that their inhibition during inflammation or infection, albeit acting via different mechanisms, may be critical in shaping the host-microbial homeostasis in the gut and affect mucosal immune responses during IBD. Development of new pharmacological approaches to either prevention of inhibition of the two apical NHEs, or restoration of their activity, may be of significant benefit to IBD patients not only as means of reducing episodes of diarrhea, but possibly as means of modulating the gut microbiome and host’s inflammatory response.

4. Therapeutic targeting of intestinal Na⁺/H⁺ exchange

Potential for modulation of intestinal fluid and Na⁺ absorption in diarrheal and inflammatory conditions by targeting NHE3 activity

As described earlier in this review, part of the success of oral rehydration therapy and its modifications relies on the modulation of electroneutral epithelial Na⁺/H⁺ exchange. Considering the role of the C-terminus of the NHE3 protein in forming regulatory complexes responsible for NHE3 endocytic retrieval,¹⁸ Zachos et al.¹⁵³ postulated that a peptide designed to mimic the region of the NHE3 protein involved in the formation of the Inhibitor Regulatory Complex (IRCX; NHERFs 1–4, PLC-γ, CK2, and CaM kinase II) may increase the apical pool of NHE3 and improve its activity. This 38-amino acid decoy peptide dubbed IRCX-CP stimulated basal NHE3 activity by 40% and prevented inhibition of NHE3 activity by both forskolin and carbachol, without altering the stimulatory effect of EGF on NHE3 activity.¹⁵³ Additionally, in vivo, IRCX-CP prevented the cholera toxin-induced increase in luminal fluid accumulation. It remains to be seen whether these promising data translate into a viable treatment. However, it clearly shows that the formation of the regulatory complexes at the C-terminus of NHE3 represents an attractive target for compound screening and development of drugs to stimulate NHE3 as potentially useful in the treatment of infectious and non-infectious diarrhea, and perhaps in modulating microbial and inflammatory responses in IBD.

Inhibition of intestinal NHE3 activity as a treatment modality for hypertension, constipation-predominant irritable bowel syndrome (IBS-C), and hyperphosphatemia for end-stage renal disease (ESRD) patients on dialysis

On the opposite spectrum, excessive sodium intake and/or reduced excretion in the intestine leads to sodium-fluid imbalances that result in hypertension, which can then lead to vision impairment in addition to a variety of cardiovascular and renal diseases such as heart failure, chronic kidney disease, stroke, and coronary heart disease. Typically, hypertension is considered to be one of the most common modifiable risk factor for cardiovascular disease and death, ¹⁵⁴ as it can be controlled by reducing dietary sodium intake.¹⁵⁵ Unfortunately, compliance with a low-salt diet is low.¹⁵⁶ Drugs that limit intestinal sodium absorption, given alone or in conjunction with other antihypertensive medication, have been considered. To this end, two oral non-absorbable NHE3 inhibitors have been developed, SAR218034 (SAR) and Tenapanor. Pharmacokinetic analyses in rats, dogs, and humans demonstrated that neither of these compounds crossed the intestinal barrier in biologically active doses,^{157, 158} and Tenapanor was well tolerated in phase I clinical study.¹⁵⁹ Both drugs were found to increase fecal and reduce urinary Na⁺ concentrations in rats (SAR and Tenapanor) and humans (Tenapanor). Not surprisingly, both drugs caused an increase in luminal fluid resulting from elevated Na⁺, leading to loose stools (a laxative effect). ^{157, 158} In a spontaneously hypertensive rat model, SAR administration in conjunction with sodium chloride laden drinking water markedly reduced systolic blood pressure.¹⁵⁸ Furthermore, in a salt-fed 5/6^th nephrectomized rat model (a Chronic Kidney Disease model associated with hypertension, hypervolemia, cardiac hypertrophy, and arterial stiffening), Tenapanor reduced extracellular volume expansion, albuminuria, and blood pressure, in addition to promoting protective cardiorenal effects such as reducing left ventricular hypertrophy.¹⁵⁷ Both drugs exhibit enhanced effects if administered in conjunction with an ACE-inhibitor, which was deemed important in cases where hypertension cannot be controlled by the administration of a single medication. ^{157, 158} However, more recently, the development of Tenapanor and SAR, and the general concept of inhibition of intestinal Na⁺/H⁺ exchange as an antihypertensive strategy appears to have been abandoned by Ardelyx and Sanofi, respectively. Ardelyx continues the investigation into the use of Tenapanor and its close analogs in patients with constipation-predominant irritable bowel syndrome (IBS-C) and for the treatment of hyperphosphatemia in ESRD patients on dialysis.^{160, 161} The potential of NHE3 inhibitors as stool softeners in IBS-C is obvious, though it remains to be seen if they can be applied without significant adverse effects. A somewhat similar strategy to modulate ion transport in constipation has been recently applied to develop CFTR activator to increase the net chloride output in the gut.¹⁶² The concept behind the use of NHE3 inhibition in hyperphosphatemia is based on the described increased fecal P_i excretion and reduced urinary P excretion in NHE3 inhibitor-treated rats with chronic kidney disease with vascular calcification, where Tenapanor markedly reduced ectopic calcification and protected renal function.¹⁶¹ The mechanism of this phenomenon remains unclear. It is also not yet evident whether targeting NHE3 would be more efficacious and with less adverse effects that the P_i binders currently used clinically.

CONCLUSIONS

NHEs are important regulators of a wide array of processes on a cellular, tissue, and systemic levels. The majority of current knowledge is limited to the function of plasmalemmal isoforms, which play protective roles during disease pathogenesis in a segment- and isoform-specific fashions and are frequent targets of inhibition by microbial or inflammatory stimuli. As such, future studies may bring new approaches that selectively target NHEs in the gut to enhance or inhibit their function for the benefit of the patients. Importantly, a large body of knowledge relies on studies focused on individual isoforms, frequently with knockout mice, while their true function always occurs in the larger context of frequently inter-dependent transporters, also subject to regulation during physiological and pathophysiological conditions. Thus, more integrative approaches to function, regulation, and targeting of NHEs in gut-associated pathologies may be needed. While the usefulness of orally available inhibitors remains to be established, the emerging focus on development of agonists of intestinal apical Na⁺/H⁺ exchange may provide significant benefits to the future treatment of infectious and inflammatory disorders of the gut.

Figure 2.

Summary graph illustrating the roles of NHE3 and NHE8 and the consequences of their inhibition during mucosal inflammation.

SUMMARY.

Epithelial Na⁺/H⁺ exchange (NHE) is a pleiotropic membrane transport mechanism which participates in the intestinal NaCl transport, in the regulation of basic cellular functions and the extracellular milieu to facilitate other nutrient absorption and to regulate gut microbial microenvironment. In this review, we summarize the findings describing the basic roles of NHE and the consequences of its dysregulation in the pathogenesis of gastrointestinal disorders.

Abbreviations used in this paper

β-PIX

Rho Guanine Nucleotide Exchange Factor 7

CaM kinase II

Ca²⁺/Calmodulin-dependent Protein Kinase II

cAMP

Cyclic Adenosine Monophosphate

CD

Crohn’s Disease

CFTR

Cystic Fibrosis Transmembrane Conductance Regulator

cGMP

Cyclic Guanosine Monophosphate

cGMPKII

Cyclic Guanosine Monophosphate Kinase II

CK2

casein kinase 2

CPA

Cation/Proton Antiporter

CSD

Congenital Sodium Diarrhea

DAEC

diffusely adherent E. coli

DRA

Downregulated-in-Adenoma

DSS

Dextran Sulfate Sodium

EGF

Epidermal Growth Factor

EAEC

enteroaggregative E. coli

EHEC

enterohemorrhagic E. coli

EPEC

Enteropathogenic E. coli

ERSD

End-Stage Renal Disease

ETEC

enterotoxigenic E. coli

GTP

Guanosine Triphosphate

GUCY2C

guanylate cyclase c gene

HGF

Human Gastric Myofibroblast

IBD

Inflammatory Bowel Disease

IBS-C

Irritable Bowel Syndrome with Constipation

IFG-II

Insulin-like Growth Factor

IFN-γ

Interferon gamma

IL-2

Interleukin 2

IL-8

Interleukin 8

IL-10

Interleukin 10

IRCX

Inhibitor Regulatory Complex

IRCX-CP

IRCX Complex Formation

mTOR

Mechanistic or Mammalian Target of Rapamycin

mTORC1

mTOR Complex 1

mTORC2

mTOR Complex 2

Muc2

Mucin 2

NaT-DC

Na⁺-transporting carboxylic acid decarboxylase

NHERF

Sodium Hydrogen Exchange Regulatory Factor

NHA

Na⁺/H⁺ Antiporter

NHE

Na⁺/H⁺ Exchange

NKA

Na⁺/K⁺-ATPase

ORS

Oral Rehydration Solution

PDZ

structural domain [combines the first letters of the following proteins - Post Synaptic Density Protein (PSD95), Drosophila Disc Large Tumor Suppressor (DLG1), and Zona Occludens-1 Protein (ZO-1)]

PDZK1

Sodium Hydrogen Exchange Regulatory Factor 3

pH_i

intracellular pH

PKC

Protein Kinase C

PLC-γ

Phospholipase C-γ

PSE

Pho1 phosphate permease

SAR

SAR218034

SCFA

Short-Chain Fatty Acids

Shank2

SH3 and Multiple Ankyrin Repeat Domains 2

Th2

T helper 2

TNBS

Trinitrobenzene sulfonic acid

TxB

C. difficile Toxin B

UC

Ulcerative Colitis