Sunil Yeruva

Lysophosphatidic Acid Stimulates the Intestinal Brush Border Na+/H+ Exchanger 3 and Fluid Absorption via LPA5 and NHERF2

BACKGROUND & AIMS Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na(+)/H(+) exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na(+)/H(+) exchanger activity and Na(+)-dependent fluid absorption in the intestine. METHODS We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na(+)/H(+) exchanger regulatory factor 2 (NHERF2; Nherf2(-/-)) or LPA(2) (Lpa(2)(-/-)). Roles of LPA(5) and NHERF2 were determined by analysis of heterologous expression. RESULTS Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA(5) was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA(5) signaling required coexpression of NHERF2, which interacted with LPA(5). LPA-mediated intestinal fluid absorption was impaired in Nherf2(-/-) mice, demonstrating the requirement for NHERF2 in LPA(5) activity. However, fluid absorption was unaltered in Lpa(2)(-/-) mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. CONCLUSIONS LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA(5). Regulation by LPA(5) depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.

Loss of downregulated in adenoma (DRA) impairs mucosal HCO3− secretion in murine ileocolonic inflammation

Background: Ileocolonic luminal pH has been reported to be abnormally low in inflammatory bowel disease (IBD) patients, and one of the causative factors may be reduced epithelial HCOSymbol secretory rate (JSymbol). Disturbances in JSymbol may occur due to inflammation‐induced changes in the crypt and villous architecture, or due to the effect of proinflammatory cytokines on epithelial ion transporters. Symbol. No caption available. Symbol. No caption available. Symbol. No caption available. Methods: To discriminate between these possibilities, the tumor necrosis factor alpha (TNF‐&agr;) overexpressing (TNF+/&Dgr;ARE) mouse model was chosen, which displays high proinflammatory cytokine levels in both ileum and colon, but develops only mild colonic histopathology and diarrhea. HCOSymbol secretion, mRNA expression, immunohistochemistry, and fluid absorptive capacity were measured in ileal and mid‐colonic mucosa of TNF+/&Dgr;ARE and wildtype (WT) (TNF+/+) mice in Ussing chambers, and in anesthetized mice in vivo. Symbol. No caption available. Results: The high basal JSymbol observed in WT ileal and mid‐colonic mucosa were luminal Cl−‐dependent and strongly decreased in TNF+/&Dgr;ARE mice. Downregulated in adenoma (DRA) mRNA and protein expression was strongly decreased in TNF+/&Dgr;ARE ileocolon, whereas cystic fibrosis transmembrane conductance regulator (CFTR), Na+/H+ exchanger 3 (NHE3), Na+/HCOSymbol cotransporter (NBC), and epithelial sodium channel (ENaC) expression was not significantly altered. This indicates that the severe defect in ileocolonic JSymbol was due to DRA downregulation. Fluid absorption was severely depressed in the ileum but only mildly affected in the mid‐distal colon, preventing the development of overt diarrhea. Symbol. No caption available. Symbol. No caption available. Symbol. No caption available. Conclusions: Even mild ileocolonic inflammation may result in a decrease of epithelial HCOSymbol secretion, which may contribute to alterations in surface pH, intestinal flora, and mucus barrier properties. (Inflamm Bowel Dis 2011;) Symbol. No caption available.

Preserved Na(+)/H(+) exchanger isoform 3 expression and localization, but decreased NHE3 function indicate regulatory sodium transport defect in ulcerative colitis.

Background: A major causative factor of diarrhea in ulcerative colitis (UC) patients is the loss of Na+ absorptive capacity of the inflamed colonic mucosa. Potential contributing mechanisms include reduced driving force for active transport, and impaired expression, mislocalization, or defective transport function of Na+ absorptive proteins. We therefore studied the expression, brush border membrane (BBM) localization, and transport capacity of the major intestinal Na+ absorptive protein, the Na+/H+ exchanger isoform 3 (NHE3) in biopsies from UC patients. Methods: In UC and control biopsies, inflammation was graded histologically, NHE3, tumor necrosis factor alpha (TNF‐&agr;), villin, as well as other housekeeping genes were analyzed by quantitative real‐time polymerase chain reaction (PCR), BBM localization of NHE3 determined by immunohistochemistry, and confocal microscopy. Na+ absorptive capacity was assessed by 22Na+ isotope fluxes and NHE3 transport activity measured microfluorometrically in BCECF‐loaded surface colonocytes within isolated crypts. Results: In mildly, moderately, and severely inflamed sigmoid colon of UC patients, neither NHE3 mRNA expression nor the abundance of NHE3 in the BBM was significantly altered compared to other structural components of the BBM. However, Na+ absorption was strongly reduced by ≈80% and acid‐activated NHE3 transport activity was significantly decreased in the surface cells of sigmoid colonic crypts even in moderately inflamed mucosa. Conclusions: In the colonic mucosa of patients with active UC, NHE3 transport capacity was found significantly decreased despite correct NHE3 location and abundance in the brush border, independent of current treatment. These findings suggest functional NHE3 transport as a novel factor for inflammatory diarrhea in UC patients. (Inflamm Bowel Dis 2010)

New therapeutic targets in ulcerative colitis: The importance of ion transporters in the human colon

Background: The absorption of water and ions (especially Na+ and Cl−) is an important function of colonic epithelial cells in both physiological and pathophysiological conditions. Despite the comprehensive animal studies, there are only scarce available data on the ion transporter activities of the normal and inflamed human colon. Methods: In this study, 128 healthy controls and 69 patients suffering from ulcerative colitis (UC) were involved. We investigated the expressional and functional characteristics of the Na+/H+ exchangers (NHE) 1–3, the epithelial sodium channel (ENaC), and the SLC26A3 Cl−/HCOSymbol exchanger downregulated in adenoma (DRA) in primary colonic crypts isolated from human biopsy and surgical samples using microfluorometry, patch clamp, and real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) techniques. Symbol. No caption available. Results: Data collected from colonic crypts showed that the activities of electroneutral (via NHE3) and the electrogenic Na+ absorption (via ENaC) are in inverse ratio to each other in the proximal and distal colon. We found no significant differences in the activity of NHE2 in different segments of the colon. Surface cell Cl−/HCOSymbol exchange is more active in the distal part of the colon. Importantly, both sodium and chloride absorptions are damaged in UC, whereas NHE1, which has been shown to promote immune response, is upregulated by 6‐fold. Symbol. No caption available. Conclusions: These results open up new therapeutic targets in UC. (Inflamm Bowel Dis 2011;)

Gene ablation for PEPT1 in mice abolishes the effects of dipeptides on small intestinal fluid absorption, short-circuit current, and intracellular pH

PEPT1 function in mouse intestine has not been assessed by means of electrophysiology and methods to assess its role in intracellular pH and fluid homeostasis. Therefore, the effects of the dipeptide glycilsarcosin (Gly-Sar) on jejunal fluid absorption and villous enterocyte intracellular pH (pH(i)) in vivo, as well as on enterocyte[(14)C]Gly-Sar uptake, short-circuit current (I(sc)) response, and enterocyte pH(i) in vitro were determined in wild-type and PEPT1-deficient mice and in mice lacking PEPT1. Immunohistochemistry for PEPT1 failed to detect any protein in enterocyte apical membranes in Slc15a1(-/-) animals. Saturable Gly-Sar uptake in Slc15a1(-/-) everted sac preparations was no longer detectable. Similarly, Gly-Sar-induced jejunal I(sc) response in vitro was abolished. The dipeptide-induced increase in fluid absorption in vivo was also abolished in animals lacking PEPT1. Since PEPT1 acts as an acid loader in enterocytes, enterocyte pH(i) was measured in vivo by two-photon microscopy in SNARF-4-loaded villous enterocytes of exteriorized jejuni in anesthetized mice, as well as in BCECF-loaded enterocytes of microdissected jejunal villi. Gly-Sar-induced pH(i) decrease was no longer observed in the absence of PEPT1. A reversal of the proton gradient across the luminal membrane did not significantly diminish Gly-Sar-induced I(sc) response, whereas a depolarization of the apical membrane potential by high K(+) or via Na(+)-K(+)-ATPase inhibition strongly diminished Gly-Sar-induced I(sc) responses. This study demonstrates for the first time that proton-coupled electrogenic dipeptide uptake in the native small intestine, mediated by PEPT1, relies on the negative apical membrane potential as the major driving force and contributes significantly to intestinal fluid transport.

Loss of PDZ‐adaptor protein NHERF2 affects membrane localization and cGMP‐ and [Ca2+]‐ but not cAMP‐dependent regulation of Na+/H+ exchanger 3 in murine intestine

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal‐dependent and possibly segment‐specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger‐mediated and receptor‐mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid‐activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin‐induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore‐ or carbachol‐mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal‐ and segment‐specific fashion, and is therefore an important regulator of intestinal fluid transport.

Knockout mouse models for intestinal electrolyte transporters and regulatory PDZ adaptors: new insights into cystic fibrosis, secretory diarrhoea and fructose-induced hypertension.

Knockout mouse models have provided key insights into the physiological significance of many intestinal electrolyte transporters. This review has selected three examples to highlight the importance of knockout mouse technology in unravelling complex regulatory relationships important for the understanding of human diseases. Genetic ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) has created one of the most useful mouse models for understanding intestinal transport. Recent work has provided an understanding of the key role of the CFTR anion channel in the regulation of HCO3− secretion, and the important consequences that a defect in HCO3− output may have on the viscoelastic properties of mucus, on lipid absorption and on male and female reproductive function. The regulation of CFTR activity, and also that of the intestinal salt absorptive transporter NHE3, occurs via the formation of PSD95‐Drosophila homologue Discs‐large‐tight junction protein ZO‐1 (PDZ) adaptor protein‐mediated multiprotein complexes. The recent generation of knockout mice for three members of the sodium‐hydrogen regulatory factor (NHERF) family of PDZ adaptor proteins, namely NHERF1 (EBP50), NHERF2 (E3KARP) and NHERF3 (PDZK1), has helped to explain why NHERF1 is essential for both normal and mutant CFTR function. In addition, they have provided new insight into the molecular mechanisms of secretory diarrhoeas. Genetic ablation of members of the recently discovered Slc26 anion transporter gene family not only reproduced the phenotype of the genetic diseases that led to the discovery of the gene family, but also resulted in new insights into complex human diseases such as secretory diarrhoea, fructose‐induced hypertension and urolithiasis.

Loss of Slc26a9 anion transporter alters intestinal electrolyte and HCO3- transport and reduces survival in CFTR-deficient mice

Slc26a9 is an anion transporter that is strongly expressed in the stomach and lung. Slc26a9 variants were recently found associated with a higher incidence of meconium ileus in cystic fibrosis (CF) infants, raising the question whether Slc26a9 is expressed in the intestine and what its functional role is. Slc26a9 messenger RNA (mRNA) was found highly expressed in the mucosae of the murine and human upper gastrointestinal tract, with an abrupt decrease in expression levels beyond the duodenum. Absence of SLC26a9 expression strongly increased the intestinally related mortality in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice. Proximal duodenal JHCO3− and fluid secretion were reduced in the absence of Slc26a9 expression. In the proximal duodenum of young Slc26a9 KO mice, the glands and villi/crypts were elongated and proliferation was enhanced. This difference was lost with ageing, as were the alterations in fluid movement, whereas the reduction in JHCO3- remained. Laser dissection followed by qPCR suggested Slc26a9 expression to be crypt-predominant in the duodenum. In summary, deletion of Slc26a9 caused bicarbonate secretory and fluid absorptive changes in the proximal duodenal mucosa and increased the postweaning death rates in CFTR-deficient mice. Functional alterations in the duodenum were most prominent at young ages. We assume that the association of meconium ileus and Slc26a9 variants may be related to maldigestion and impaired downstream signaling caused by loss of upper GI tract digestive functions, aggravating the situation of lack of secretion and sticky mucus at the site of obstruction in CF intestine.

Evidence for a causal link between adaptor protein PDZK1 downregulation and Na + /H + exchanger NHE3 dysfunction in human and murine colitis

A dysfunction of the Na+/H+ exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

Contributions of intestinal epithelial barriers to health and disease

A core function of epithelia is to form barriers that separate tissue compartments within complex organisms. These barriers also separate the internal milieu from the external environment and are, therefore, an essential component of host defense. However, in many cases, a perfect barrier would be improbable with life itself. Examples include the air spaces within the lungs, the renal tubules, and the intestines. Here, we focus on the mechanisms by which barriers are assembled, maintained, and regulated in the context of health and disease. Because of its unique challenges and extensive study, we focus on the gastrointestinal tract as an organ-specific example of the essential contributions of the paracellular barrier to life.