DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Dietary Cinnamaldehyde Activation of TRPA1 Antagonizes High-Salt-Induced Hypertension Through Restoring Renal Tubular Mitochondrial Dysfunction.

BACKGROUND: The renal proximal tubule (RPT) plays a pivotal role in regulating sodium reabsorption and thus blood pressure (BP). Transient receptor potential ankyrin 1 (TRPA1) has been reported to protect against renal injury by modulating mitochondrial function. We hypothesize that the activation of TRPA1 by its agonist cinnamaldehyde may mitigate high-salt intake-induced hypertension by inhibiting urinary sodium reabsorption through restoration of renal tubular epithelial mitochondrial function. METHODS: Trpa1-deficient (Trpa1-/-) mice and wild-type (WT) mice were fed standard laboratory chow [normal diet (ND) group, 0.4% salt], standard laboratory chow with 8% salt [high-salt diet (HS) group], or standard laboratory chow with 8% salt plus 0.015% cinnamaldehyde [high-salt plus cinnamaldehyde diet (HSC) group] for 6 months. Urinary sodium excretion, reactive oxygen species (ROS) production, mitochondrial function, and the expression of sodium hydrogen exchanger isoform 3 (NHE3) and Na+/K+-ATPase of RPTs were determined. RESULTS: Chronic dietary cinnamaldehyde supplementation reduced tail systolic BP and 24-hour ambulatory arterial pressure in HS-fed WT mice. Compared with the mice fed HS, cinnamaldehyde supplementation significantly increased urinary sodium excretion, inhibited excess ROS production, and alleviated mitochondrial dysfunction of RPTs in WT mice. However, these effects of cinnamaldehyde were absent in Trpa1-/- mice. Furthermore, chronic dietary cinnamaldehyde supplementation blunted HS-induced upregulation of NHE3 and Na+/K+-ATPase in WT mice but not Trpa1-/- mice. CONCLUSIONS: The present study demonstrated that chronic activation of Trpa1 attenuates HS-induced hypertension by inhibiting urinary sodium reabsorption through restoring renal tubular epithelial mitochondrial function. Renal TRPA1 may be a potential target for the management of excessive dietary salt intake-associated hypertension.

Porcine delta coronavirus inhibits NHE3 activity of porcine intestinal epithelial cells through miR-361-3p/NHE3 regulatory axis.

Porcine deltacoronavirus (PDCoV) infection in piglets can cause small intestinal epithelial necrosis and atrophic enteritis, which leads to severe damages to host cells, and result in diarrhea. In this study, we investigated the relationship between miR-361, SLC9A3(Solute carrier family 9, subfamily A, member 3), and NHE3(sodium-hydrogen exchanger member 3) in in porcine intestinal epithelial cells (IPI-2I) cells after PDCoV infection. Our results showed that the ssc-miR-361-3p expression inhibits the mRNA level of SLC9A3 gene which lead to the descending of NHE3 protein expression, and the NHE3 activity was suppressed. NHE3 activity was suppressed via down-regulation expression of SLC9A3 mRNA by transfection with siRNA. Ssc-miR-361-3p mimics and inhibitors were used to change the expression of ssc-miR-361-3p in IPI-2I cells. Ssc-miR-361-3p overexpression reduced the mRNA level of SLC9A3 gene, the level of NHE3 protein expression and NHE3 activity in IPI-2I cells, while ssc-miR-361-3p inhibits NHE3. Furthermore, luciferase reporter assay showed that SLC9A3 gene was a direct target of ssc-miR-361-3p. Ssc-miR-361-3p inhibition restored NHE3 activity in PDCoV infected IPI-2I cells by up-regulating SLC9A3 mRNA expression and NHE3 protein expression. These results demonstrate that the PDCoV infection can inhibit NHE3 activity through miR-361-3p/SLC9A3 regulatory axis. The relevant research is reported for the first time in PDCoV, which has significance in exploring the pathogenic mechanism of PDCoV and can provide a theoretical basis for its prevention and control. suggesting that NHE3 and ssc-miR-361-3p may be potential therapeutic targets for diarrhea in infected piglets.

A Novel Peptide Prevents Enterotoxin- and Inflammation-Induced Intestinal Fluid Secretion by Stimulating Sodium-Hydrogen Exchanger 3 Activity.

BACKGROUND & AIMS: Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. METHODS: A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine in vitro and in vivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. RESULTS: N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and Ca2+ in cell lines and in in vitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine in vivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. CONCLUSIONS: These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.

SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+-dependent manner by cAMP and ATP in intestinal epithelial cells.

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl-/[Formula: see text] exchanger that is part of neutral NaCl absorption under baseline conditions, but in cyclic adenosine monophosphate (cAMP)-driven diarrheas, it is stimulated and contributes to increased anion secretion. To further understand the regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and adenosine 5'-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 µM and ATP at 0.25 µM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP increased intracellular Ca2+ (Ca2+i) in a concentration-dependent manner, whereas FSK (1 µM), which by itself did not significantly alter Ca2+i, followed by 0.25 µM ATP produced a large increase in Ca2+ that was approximately equal to the elevation caused by 1 µM ATP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) pretreatment prevented the ATP and FSK/ATP synergistically increased the DRA activity and the increase in Ca2+i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca2+) synergistically increased Ca2+i and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases, such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, whereas separation of DRA from Na+/H+ exchanger isoform-3 (NHE3) contributes to reduced NaCl absorption.NEW & NOTEWORTHY The BB Cl-/[Formula: see text] exchanger DRA takes part in both neutral NaCl absorption and stimulated anion secretion. Using intestinal cell line, Caco-2/BBE high concentrations of cAMP and Ca2+ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca2+. This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca2+ are involved.

Enhanced phosphate absorption in intestinal epithelial cell-specific NHE3 knockout mice.

AIMS: The kidneys play a major role in maintaining Pi homeostasis. Patients in later stages of CKD develop hyperphosphatemia. One novel treatment option is tenapanor, an intestinal-specific NHE3 inhibitor. To gain mechanistic insight into the role of intestinal NHE3 in Pi homeostasis, we studied tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout (NHE3IEC-KO ) mice. METHODS: Mice underwent dietary Pi challenges, and hormones as well as urinary/plasma Pi were determined. Intestinal 33 P uptake studies were conducted in vivo to compare the effects of tenapanor and NHE3IEC-KO . Ex vivo Pi transport was measured in everted gut sacs and brush border membrane vesicles. Intestinal and renal protein expression of Pi transporters were determined. RESULTS: On the control diet, NHE3IEC-KO mice had similar Pi homeostasis, but a ~25% reduction in FGF23 compared with control mice. Everted gut sacs and brush border membrane vesicles showed enhanced Pi uptake associated with increased Npt2b expression in NHE3IEC-KO mice. Acute oral Pi loading resulted in higher plasma Pi in NHE3IEC-KO mice. Tenapanor inhibited intestinal 33 P uptake acutely but then led to hyper-absorption at later time points compared to vehicle. In response to high dietary Pi , plasma Pi and FGF23 increased to higher levels in NHE3IEC-KO mice which was associated with greater Npt2b expression. Reduced renal Npt2c and a trend for reduced Npt2a expression were unable to correct for higher plasma Pi . CONCLUSION: Intestinal NHE3 has a significant contribution to Pi homeostasis. In contrast to effects described for tenapanor on Pi homeostasis, NHE3IEC-KO mice show enhanced, rather than reduced, intestinal Pi uptake.

Adaptive changes in single-nephron GFR, tubular morphology, and transport in a pregnant rat nephron: modeling and analysis.

Normal pregnancy is characterized by massive increases in plasma volume and electrolyte retention. Given that the kidneys regulate homeostasis of electrolytes and volume, the organ undergoes major adaptations in morphology, hemodynamics, and transport to achieve the volume and electrolyte retention required in pregnancy. These adaptations are complex, sometimes counterintuitive, and not fully understood. In addition, the demands of the developing fetus and placenta change throughout pregnancy. For example, during late pregnancy, K+ retention and thus enhanced renal K+ reabsorption are required despite many kaliuretic factors. The goal of this study was to unravel how known adaptive changes along the nephrons contribute to the ability of the kidney to meet volume and electrolyte requirements in mid and late pregnancy. We developed computational models of solute and water transport in the superficial nephron of the kidney of a rat in mid and late pregnancy. The midpregnant and late-pregnant rat superficial nephron models predicted that morphological adaptations and increased activity of Na+/H+ exchanger 3 (NHE3) and epithelial Na+ channel are essential for the enhanced Na+ reabsorption observed during pregnancy. Model simulations showed that for sufficient K+ reabsorption, increased activity of H+-K+-ATPase and decreased K+ secretion along the distal segments is required in both mid and late pregnancy. The model results also suggested that certain known sex differences in renal transporter pattern (e.g., the higher NHE3 protein abundance but lower activity in the proximal tubules of virgin female rats compared with male rats) may serve to better prepare females for the increased transport demand in pregnancy.NEW & NOTEWORTHY Normal pregnancy in mammals is generally characterized by massive changes in plasma volume and electrolyte retention. This study provides insights into how the volume and electrolyte requirement in different pregnancy stages are met by coordinated adaptive changes in the kidney. The model results also suggested that certain known sex differences in the renal transporter pattern may serve to better prepare females for the increased transport demand in pregnancy.

Decreased NHE3 activity in intestinal epithelial cells in TGEV and PEDV-induced piglet diarrhea.

Transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED) are highly transmissible intestinal infections caused by transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV), respectively. They are clinically associated with vomiting, diarrhea, and dehydration in piglets. An imbalance in Na+ uptake by intestinal epithelial cells causes TGEV/PEDV-induced diarrhea. However, the mechanism by which TGEV/PEDV-infection in piglets causes Na+ imbalance diarrhea has not been elucidated. In the present study, we demonstrated that specific inhibition of NHE3 activity caused small intestinal bulging, intestinal wall thinning and severe diarrhea in piglets, consistent with the signs of TGEV/PEDV infection. This study further elucidated the role of NHE3 in TGEV/PEDV-induced diarrhea. In this study, small intestinal epithelial cells (IPEC-J2) were used as a model of infection. The results showed that TGEV/PEDV infection reduced NHE3 activity and Na+ uptake in IPEC-J2 cells. Further studies revealed that the use of NHE3-specific inhibitors could reduce the amount of cell membrane NHE3, thereby decreasing Na+ uptake and ultimately leading to diarrhea. Transcriptomic studies performed on obtained jejunal tissues were also consistent with pre-laboratory results. This study will provide a basis for understanding Na+ imbalance diarrhea caused by TGEV/PEDV, as well as for elucidating the diarrheal pathogenesis of other members of α-animal coronaviruses.

The recycling regulation of sodium-hydrogen exchanger isoform 3(NHE3) in epithelial cells.

As the main exchanger of electroneutral NaCl absorption, sodium-hydrogen exchanger isoform 3 (NHE3) circulates in the epithelial brush border (BB) and intracellular compartments in a multi-protein complex. The size of the NHE3 complex changes during rapid regulation events. Recycling regulation of NHE3 in epithelial cells can be roughly divided into three stages. First, when stimulated by Ca2+, cGMP, and cAMP-dependent signaling pathways, NHE3 is converted from an immobile complex found at the apical microvilli (MV) into an easily internalized and mobile form that relocates to a compartment near the base of the MV. Second, NHE3 is internalized by clathrin and albumin-dependent pathways into cytoplasmic endosomal compartments, where the complex is reprocessed and reassembled. Finally, NHE3 is translocated from the recycling endosomes (REs) to the apex of epithelial cells, a process that can be stimulated by an increase in sodium-glucose cotransporter 1 (SGLT1) activity, epidermal growth factor receptor (EGFR) signaling, Ca2+ signaling, and binding to ßPix and SH3 and multiple ankyrin repeat domains 2 (Shank2) proteins. This review describes the molecular steps and protein interactions involved in the recycling movement of NHE3 from the apex of epithelial cells, into vesicles, where it is reprocessed and reassembled, and returned to its original location on the plasma membrane, where it exerts its physiological function.

Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle.

Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl- (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.

The role of NHE3 (Slc9a3) in oxalate and sodium transport by mouse intestine and regulation by cAMP.

Intestinal oxalate transport involves Cl- /HCO3- exchangers but how this transport is regulated is not currently known. NHE3 (Slc9a3), an apical Na+ /H+ exchanger, is an established target for regulation of electroneutral NaCl absorption working in concert with Cl- /HCO3- exchangers. To test whether NHE3 could be involved in regulation of intestinal oxalate transport and renal oxalate handling we compared urinary oxalate excretion rates and intestinal transepithelial fluxes of 14 C-oxalate and 22 Na+ between NHE3 KO and wild-type (WT) mice. NHE3 KO kidneys had lower creatinine clearance suggesting reduced GFR, but urinary oxalate excretion rates (µmol/24 h) were similar compared to the WT but doubled when expressed as a ratio of creatinine. Intestinal transepithelial fluxes of 14 C-oxalate and 22 Na+ were measured in the distal ileum, cecum, and distal colon. The absence of NHE3 did not affect basal net transport rates of oxalate or sodium across any intestinal section examined. Stimulation of intracellular cAMP with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) led to an increase in net oxalate secretion in the WT distal ileum and cecum and inhibition of sodium absorption in the cecum and distal colon. In NHE3 KO cecum, cAMP stimulation of oxalate secretion was impaired suggesting the possibility of a role for NHE3 in this process. Although, there is little evidence for a role of NHE3 in basal intestinal oxalate fluxes, NHE3 may be important for cAMP stimulation of oxalate in the cecum and for renal handling of oxalate.

Inhibition of Na+ /H+ exchanger isoform 3 improves gut fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator-deficient and F508del mutant mice.

BACKGROUND AND PURPOSE: Constipation and intestinal obstructive episodes are major health problems in cystic fibrosis (CF) patients. Three FDA-approved drugs against constipation-prone irritable bowel syndrome were tested for their ability to increase luminal fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator (CFTR) null (cftr-/- ) and F508del mutant (F508delmut/mut ) murine intestine. EXPERIMENTAL APPROACH: Guanylate cyclase C agonist linaclotide, PGE1 analogue lubiprostone and intestine-specific NHE3 inhibitor tenapanor were perfused through a ~3 cm jejunal, proximal or mid-distal colonic segment in anaesthetized cftr-/- , F508delmut/mut and WT mice. Net fluid balance was determined gravimetrically and alkaline output by pH-stat back titration. KEY RESULTS: Basal jejunal fluid absorptive rates were significantly higher and basal HCO3- output was significantly lower in cftr-/- and F508delmut/mut compared to WT mice. In cftr-/- and F508delmut/mut mice, all three drugs significantly inhibited the fluid absorptive rate and increased alkaline output in the jejunum and tenapanor and lubiprostone, but not linaclotide, in the colon. After tenapanor pre-incubation, linaclotide elicited a robust fluid secretory response in WT jejunum, while no further change in absorptive rates was observed in cftr-/- and F508delmut/mut jejunum, suggesting that the increase in gut fluidity and alkalinity by linaclotide in CF gut is mediated via NHE3 inhibition. Lubiprostone also inhibited fluid absorption in cftr-/- and F508delmut/mut jejunum via NHE3 inhibition but had a residual NHE3-independent effect. CONCLUSION AND IMPLICATIONS: Linaclotide, lubiprostone and tenapanor reduced fluid absorption and increased alkaline output in the CF gut. Their application may ameliorate constipation and reduce obstructive episodes in CF patients.

Ubiquitin-specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity.

Na+ /H+ exchanger NHE3 of human or primates differs from NHE3 of other animals by having a PY motif, which mediates interaction with the E3 ubiquitin (Ub) ligase Nedd4-2. Ub-conjugation of human NHE3 by Nedd4-2 regulates endocytosis of NHE3 but does not affect its cellular expression. Because Ub-conjugation is a reversible process, the aim of this study is to identify deubiquitinating enzyme (DUB) regulating the post-endosomal fate of human NHE3. Using an activity-based chemical screening, we identified ubiquitin specific protease-7 (USP7) and USP10 that bind NHE3. The roles of DUBs in regulation of NHE3 were studied by determining the effects of silencing of USP7 and USP10. Knockdown of USP7 or USP10 resulted in increased NHE3 ubiquitination and decreased NHE3 expression at the surface membrane and cellular level. The endocytic retrieval of NHE3 was promoted by depletion of USP7 or USP10, with increased association of NHE3 with Rab5a and Rab7. Inhibition of USP7 and USP10 by chemical inhibitors or knockdown had an additive effect on NHE3. In addition, NHE3 half-life was reduced accounting for decreased NHE3 protein abundance. NHE3 is inhibited by protein kinase A. Activation of PKA by forskolin decreased the binding of USP7 and USP10 to NHE3, while increasing ubiquitination of NHE3. Knockdown of USP10 had an additive effect on PKA-dependent inhibition of NHE3. These findings demonstrate that USP7 and USP10 are DUBs that regulate NHE3 ubiquitination and expression, and reveal a new mechanism of NHE3 inhibition involving DUBs.

Segment-specific effects of resveratrol on porcine small intestinal dipeptide absorption depend on the mucosal pH and are due to different mechanisms: potential roles of different transport proteins and protein kinases.

Numerous beneficial features of the polyphenol resveratrol (RSV) have been demonstrated in several tissues and cell culture models. There is also evidence, that RSV impairs intestinal nutrient transport but the underlying mechanisms are not understood. The aim of the present study was to evaluate whether RSV has also an impact on the H+-coupled transport of peptides via the peptide transporter 1 (PepT1) and to characterize RSV mediated changes in the apical abundance of nutrients transport proteins and protein kinases that may be involved. RSV decreased the H+-coupled transport of peptides in the porcine small intestines in a pH and location specific manner (jejunum vs ileum) as measured in Ussing chamber experiments. The comparison of the effects of RSV with the effects of the cAMP/PKA-activating agent forskolin indicates that different mechanisms may be responsible in the intestinal segments. Additionally, it seems that the transport of peptides and glucose in the jejunum are inhibited via the same mechanism while there might be two mechanisms involved in the ileum. Functional data and protein expression data indicate, that, besides PepT1, the activity of the Na+/H+-exchanger 3 (NHE3) may be involved. Protein kinase A (PKA) and AMP-activated kinase (AMPK) are both activated by RSV while the extracellular regulated kinase (ERK) and the serum and glucocorticoid induced kinase (SGK) are widely unaffected. Although PKA and AMPK are activated, AMPK seems not to be related to the effects of RSV. Additionally, both the functional data and the protein expression data reveal some interesting pH- and segment-specific differences.

Altered absorptive function in the gall bladder during cholesterol gallstone formation is associated with abnormal NHE3 complex formation.

Dysfunction of the Na+/H+ exchanger 3 (NHE3) contributes to the formation of cholesterol gallstones. We aimed to investigate whether NHE3 dysfunction is associated with abnormalities in NHE3 complex formation. We fed C57BL/6 mice with control or lithogenic diet and study the expression of NHE3, ezrin, and Na+/H+ exchanger regulatory factor 1 (NHERF1) in the gallbladder (GB) using RT-PCR and western blot. Immunofluorescence and immunoprecipitation were performed to investigate the interactions of NHE3 with ezrin or NHERF1. To explore the initiating factor that leads to NHE3 dysfunction, we stimulated cholangiocarcinoma cells with taurochenodeoxycholate (TCDC) and/or forskolin. The effects of TCDC on the expression of NHE3 regulatory proteins, as well as their bindings to NHE3, were detected by western blot and immunoprecipitation. Enzyme-linked immunosorbent assay was used to study the regulation of cAMP production by TCDC. The expression of NHERF1 and ezrin phosphorylation level were increased in the gallbladder epithelial cells (GBECs) of C57BL/6 mice with cholesterol gallstones. Immunofluorescence studies demonstrated that the subcellular localization of ezrin and NHERF1 were similar to that of NHE3 in GBECs. Immunoprecipitation revealed that ezrin formed macrocomplex with NHE3, which were enhanced after gallstone formation. TCDC increased forskolin-induced cAMP accumulation, and NHERF1 and PKCα expression in cholangiocarcinoma cells. Under the synergistic effect of forskolin, TCDC stimulated ezrin phosphorylation, with enhanced interaction between ezrin and NHE3. The formation of cholesterol gallstones is associated with abnormal formation of NHE3 complexes. Decreased biliary TCDC may be an initiating factor that leads to abnormal GB absorption.

Glucocorticoids and serum- and glucocorticoid-inducible kinase 1 are potent regulators of CFTR in the native intestine: implications for stress-induced diarrhea.

Nongenomic glucocorticoid (GC) and serum- and glucocorticoid-inducible kinase 1 (SGK1) signaling regulate ion transport, but CFTR has not been investigated in the intestine. We examined GC, SGK1, and phosphatidylinositol 3-kinase (PI3K) kinase signaling of CFTR ion transport in native intestine and the role of GCs on mRNA, protein, surface expression, and cyclic guanosine monophosphate (cGMP)-elicited diarrhea. Rats were treated with dexamethasone (DEXA; 2 mg/kg ip) or DMSO for 1, 4, and 24 h. Cyclic adenosine monophosphate (cAMP)-activated ion transport was examined in the presence or absence of SGK1 and PI3K inhibitors. Phosphorylation of SGK1, phosphoinositide-dependent kinase 1, and Akt kinases was confirmed by immunoblots using phosphor-specific antibodies. Tissue lysates were analyzed by mass spectrometry. CFTR and SGK1 mRNA were measured by quantitative PCR. Changes in total and surface CFTR protein were determined. The role of GC in cGMP-activated CFTR ion transport was examined. GC synergistically increased CFTR ion transport by SGK1 and PI3K signaling and increased CFTR protein without altering SGK1 or CFTR mRNA. GC induced highest levels of CFTR protein at 4 h that were associated with marked increase in surface CFTR, phosphorylation of the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-like (Nedd4-2), and 14-3-3ε, supporting their roles in surface retention and stability. Coimmunoprecipitation of CFTR, Nedd4-2, and 14-3-3ε indicated that assembly of this complex is a likely effector of the SGK and Akt pathways. Mass spectrometry identified phosphorylated peptides in relevant proteins. GC-SGK1 potently regulates CFTR in the intestine and is implicated in diarrheal disease.NEW & NOTEWORTHY This is the first study to examine the mechanisms of glucocorticoid, serum- and glucocorticoid-inducible kinase 1, and nongenomic kinase signaling of CFTR in the native intestine. We identified unique and druggable intestine-specific factors of the pathway that are targets for treating stress-induced diarrhea.

An inducible intestinal epithelial cell-specific NHE3 knockout mouse model mimicking congenital sodium diarrhea.

The sodium-hydrogen exchanger isoform 3 (NHE3, SLC9A3) is abundantly expressed in the gastrointestinal tract and is proposed to play essential roles in Na+ and fluid absorption as well as acid-base homeostasis. Mutations in the SLC9A3 gene can cause congenital sodium diarrhea (CSD). However, understanding the precise role of intestinal NHE3 has been severely hampered due to the lack of a suitable animal model. To navigate this problem and better understand the role of intestinal NHE3, we generated a tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout mouse model (NHE3IEC-KO). Before tamoxifen administration, the phenotype and blood parameters of NHE3IEC-KO were unremarkable compared with control mice. After tamoxifen administration, NHE3IEC-KO mice have undetectable levels of NHE3 in the intestine. NHE3IEC-KO mice develop watery, alkaline diarrhea in combination with a swollen small intestine, cecum and colon. The persistent diarrhea results in higher fluid intake. After 3 weeks, NHE3IEC-KO mice show a ∼25% mortality rate. The contribution of intestinal NHE3 to acid-base and Na+ homeostasis under normal conditions becomes evident in NHE3IEC-KO mice that have metabolic acidosis, lower blood bicarbonate levels, hyponatremia and hyperkalemia associated with drastically elevated plasma aldosterone levels. These results demonstrate that intestinal NHE3 has a significant contribution to acid-base, Na+ and volume homeostasis, and lack of intestinal NHE3 has consequences on intestinal structural integrity. This mouse model mimics and explains the phenotype of individuals with CSD carrying SLC9A3 mutations.

Testosterone modulates renal ammonia metabolism.

There are substantial sex differences in renal structure and ammonia metabolism that correlate with differences in expression of proteins involved in ammonia generation and transport. This study determined the role of testis-derived testosterone in these differences. We studied 4-mo-old male C57BL/6 mice 4 and 8 wk after either bilateral orchiectomy (ORCH) or sham-operated control surgery and determined the effect of testosterone replacement to reverse the effects of ORCH. Finally, we determined the cellular expression of androgen receptor (AR), testosterone's canonical target receptor. ORCH decreased kidney and proximal tubule size, and testosterone replacement reversed this effect. ORCH increased ammonia excretion in a testosterone-dependent fashion; this occurred despite similar food intake, which is the primary component of endogenous acid production. ORCH increased expression of both phosphoenolpyruvate, a major ammonia-generating protein, and Na+-K+-2Cl- cotransporter, which mediates thick ascending limb ammonia reabsorption; these changes were reversed with testosterone replacement. Orchiectomy also decreased expression of Na+/H+ exchanger isoform 3, which mediates proximal tubule ammonia secretion, in a testosterone-dependent pattern. Finally, ARs are expressed throughout the proximal tubule in both the male and female kidney. Testosterone, possibly acting through ARs, has dramatic effects on kidney and proximal tubule size and decreases ammonia excretion through its effects on several key proteins involved in ammonia metabolism.

Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters.

BACKGROUND & AIMS: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. METHODS: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs). RESULTS: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. CONCLUSIONS: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.

Decreased NHE3 activity and trafficking in TGEV-infected IPEC-J2 cells via the SGLT1-mediated P38 MAPK/AKt2 pathway.

Transmissible gastroenteritis virus (TGEV) primarily replicates in intestinal epithelial cells and causes severe damage to host cells, resulting in diarrhea. Surface NHE3 serves as the key regulatory site controlling electroneutral Na+ absorption. In this study, our results showed that the surface NHE3 content was significantly reduced following TGEV infection, whereas the total level of protein expression was not significantly changed, and NHE3 activity gradually decreased with prolonged infection time. We then inhibited SGLT1 expression by lentiviral interference and drug inhibition, respectively. Inhibition studies showed that the level of phosphorylation of the downstream key proteins, MAPKAPK-2 and EZRIN, in the SGLT1-mediated p38MAPK/AKt2 signaling pathway was significantly increased. The surface NHE3 expression was also significantly increased, and NHE3 activity was also significantly enhanced. These results demonstrate that a TGEV infection can inhibit NHE3 translocation and attenuates sodium-hydrogen exchange activity via the SGLT1-mediated p38MAPK/AKt2 signaling pathway, affecting cellular electrolyte absorption leading to diarrhea.