Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

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.

Studies with Human-Induced Pluripotent Stem Cells Reveal That CTNS Mutations Can Alter Renal Proximal Tubule Differentiation.

Cystinosis is an autosomal recessive disease resulting from mutations in ctns, which encodes for cystinosin, a proton-coupled cystine transporter that exports cystine from lysosomes. The major clinical form, infantile cystinosis, is associated with renal failure due to the malfunctioning of the renal proximal tubule (RPT). To examine the hypothesis that the malfunctioning of the cystinotic RPT arises from defective differentiation, human-induced pluripotent stem cells (hiPSCs) were generated from human dermal fibroblasts from an individual with infantile cystinosis, as well as a normal individual. The results indicate that both the cystinotic and normal hiPSCs are pluripotent and can form embryoid bodies (EBs) with the three primordial germ layers. When the normal hiPSCs were subjected to a differentiation regime that induces RPT formation, organoids containing tubules with lumens emerged that expressed distinctive RPT proteins, including villin, the Na+/H+ Exchanger (NHE) isoform 3 (NHE3), and the NHE Regulatory Factor 1 (NHERF1). The formation of tubules with lumens was less pronounced in organoids derived from cystinotic hiPSCs, although the organoids expressed villin, NHE3, and NHERF1. These observations can be attributed to an impairment in differentiation and/or by other defects which cause cystinotic RPTs to have an increased propensity to undergo apoptosis or other types of programmed cell death.

Increased intestinal permeability and downregulation of absorptive ion transporters Nhe3, Dra, and Sglt1 contribute to diarrhea during Clostridioides difficile infection.

BACKGROUND & AIM: Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced by C. difficile are the major determinants of disease. However, the pathophysiological causes of diarrhea during CDI are not well understood. Here, we investigated the effects of C. difficile toxins on paracellular permeability and apical ion transporters in the context of an acute physiological infection. METHODS: We studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Üssing chambers were used to measure paracellular permeability and ion transporter function across the intestinal tract. Infected intestinal tissues were analyzed by immunofluorescence microscopy and RNA-sequencing to uncover mechanisms of transporter dysregulation. RESULTS: Intestinal permeability was increased through the size-selective leak pathway in vivo during acute CDI in a 2-day-post infection model. Chloride secretory activity was reduced in the cecum and distal colon during infection by decreased CaCC and CFTR function, respectively. SGLT1 activity was significantly reduced in the cecum and colon, accompanied by ablated SGLT1 expression in colonocytes and increased luminal glucose concentrations. SGLT1 and DRA expression was ablated by either TcdA or TcdB during acute infection, but NHE3 was decreased in a TcdB-dependent manner. The localization of key proteins that link filamentous actin to the ion transporters in the apical plasma membrane was unchanged. However, Sglt1, Nhe3, and Dra were drastically reduced at the transcript level, implicating downregulation of ion transporters in the mechanism of diarrhea during CDI. CONCLUSIONS: CDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination likely leads to dysfunctional water and solute absorption in the large bowel, causing osmotic diarrhea. These findings provide insights into the pathophysiological mechanisms underlying diarrhea and may open novel avenues for attenuating CDI-associated diarrhea.

Collectrin (Tmem27) deficiency in proximal tubules causes hypertension in mice and a TMEM27 variant associates with blood pressure in males in a Latino cohort.

Collectrin (Tmem27), an angiotensin-converting enzyme 2 homologue, is a chaperone of amino acid transporters in the kidney and endothelium. Global collectrin knockout (KO) mice have hypertension, endothelial dysfunction, exaggerated salt sensitivity, and diminished renal blood flow. This phenotype is associated with altered nitric oxide and superoxide balance and increased proximal tubule (PT) Na+/H+ exchanger isoform 3 (NHE3) expression. Collectrin is located on the X chromosome where genome-wide association population studies have largely been excluded. In the present study, we generated PT-specific collectrin KO (PT KO) mice to determine the precise contribution of PT collectrin in blood pressure homeostasis. We also examined the association of human TMEM27 single-nucleotide polymorphisms with blood pressure traits in 11,926 Hispanic Community Health Study/Study of Latinos (HCHS/SOL) Hispanic/Latino participants. PT KO mice exhibited hypertension, and this was associated with increased baseline NHE3 expression and diminished lithium excretion. However, PT KO mice did not display exaggerated salt sensitivity or a reduction in renal blood flow compared with control mice. Furthermore, PT KO mice exhibited enhanced endothelium-mediated dilation, suggesting a compensatory response to systemic hypertension induced by deficiency of collectrin in the PT. In HCHS/SOL participants, we observed sex-specific single-nucleotide polymorphism associations with diastolic blood pressure. In conclusion, loss of collectrin in the PT is sufficient to induce hypertension, at least in part, through activation of NHE3. Importantly, our model supports the notion that altered renal blood flow may be a determining factor for salt sensitivity. Further studies are needed to investigate the role of the TMEM27 locus on blood pressure and salt sensitivity in humans.NEW & NOTEWORTHY The findings of our study are significant in several ways: 1) loss of an amino acid chaperone in the proximal tubule is sufficient to cause hypertension, 2) the results in global and proximal tubule-specific collectrin knockout mice support the notion that vascular dysfunction is required for salt sensitivity or that impaired renal tubule function causes hypertension but is not sufficient to cause salt sensitivity, and 3) our study is the first to implicate a role of collectrin in human hypertension.

Genetic Deletion of AT1a Receptor or Na+/H+ Exchanger 3 Selectively in the Proximal Tubules of the Kidney Attenuates Two-Kidney, One-Clip Goldblatt Hypertension in Mice.

The roles of angiotensin II (Ang II) AT1 (AT1a) receptors and its downstream target Na+/H+ exchanger 3 (NHE3) in the proximal tubules in the development of two-kidney, 1-clip (2K1C) Goldblatt hypertension have not been investigated previously. The present study tested the hypothesis that deletion of the AT1a receptor or NHE3 selectively in the proximal tubules of the kidney attenuates the development of 2K1C hypertension using novel mouse models with proximal tubule-specific deletion of AT1a receptors or NHE3. 2K1C Goldblatt hypertension was induced by placing a silver clip (0.12 mm) on the left renal artery for 4 weeks in adult male wild-type (WT), global Agtr1a−/−, proximal tubule (PT)-specific PT-Agtr1a−/− or PT-Nhe3−/− mice, respectively. As expected, telemetry blood pressure increased in a time-dependent manner in WT mice, reaching a maximal response by Week 3 (p < 0.01). 2K1C hypertension in WT mice was associated with increases in renin expression in the clipped kidney and decreases in the nonclipped kidney (p < 0.05). Plasma and kidney Ang II were significantly increased in WT mice with 2K1C hypertension (p < 0.05). Tubulointerstitial fibrotic responses were significantly increased in the clipped kidney (p < 0.01). Whole-body deletion of AT1a receptors completely blocked the development of 2K1C hypertension in Agtr1a−/− mice (p < 0.01 vs. WT). Likewise, proximal tubule-specific deletion of Agtr1a in PT-Agtr1a−/− mice or NHE3 in PT-Nhe3−/− mice also blocked the development of 2K1C hypertension (p < 0.01 vs. WT). Taken together, the present study provides new evidence for a critical role of proximal tubule Ang II/AT1 (AT1a)/NHE3 axis in the development of 2K1C Goldblatt hypertension.

Transmissible Gastroenteritis Virus Nucleocapsid Protein Interacts with Na+/H+ Exchanger 3 To Reduce Na+/H+ Exchanger Activity and Promote Piglet Diarrhea.

Transmissible gastroenteritis virus (TGEV) is member of the family Coronaviridae and mainly causes acute diarrhea. TGEV infection is characterized by vomiting, watery diarrhea, and severe dehydration, resulting in high mortality rates in neonatal piglets. TGEV infection symptoms are related to an imbalance of sodium absorption in small intestinal epithelial cells; however, the etiology of sodium imbalance diarrhea caused by TGEV remains unclear. In this study, we performed transcriptomic analysis of intestinal tissues from infected and healthy piglets and observed that the expression of NHE3, encoding Na+/H+ exchanger 3 (NHE3), the main exchanger of electroneutral sodium in intestinal epithelial cells, was significantly reduced upon TGEV infection. We also showed that specific inhibition of intestinal NHE3 activity could lead to the development of diarrhea in piglets. Furthermore, we revealed an interaction between TGEV N protein and NHE3 near the nucleus. The binding of TGEV N to NHE3 directly affected the expression and activity of NHE3 on the cell surface and affected cellular electrolyte absorption, leading to diarrhea. Molecular docking and computer-aided screening techniques were used to screen for the blocker of the interaction between TGEV N and NHE3, which identified irinotecan. We then demonstrated that irinotecan was effective in relieving TGEV-induced diarrhea in piglets. These findings provide new insights into the mechanism of TGEV-induced sodium imbalance diarrhea and could lead to the design of novel antiviral strategies against TGEV. IMPORTANCE A variety of coronaviruses have been found to cause severe diarrhea in hosts, including TGEV; however, the pathogenic mechanism is not clear. Therefore, prompt determination of the mechanism and identification of efficient therapeutic agents are required, both for public health reasons and for economic development. In this study, we demonstrated that NHE3 is the major expressed protein of NHEs in the intestine, and its expression decreased by nearly 70% after TGEV infection. Also, specific inhibition of intestinal NHE3 resulted in severe diarrhea in piglets. This demonstrated that NHE3 plays an important role in TGEV-induced diarrhea. In addition, we found that TGEV N directly regulates NHE3 expression and activity through protein-protein interaction, which is essential to promote diarrhea. Molecular docking and other techniques demonstrated that irinotecan could block the interaction and diarrhea caused by TGEV. Thus, our results provide a basis for the development of novel therapeutic agents against TGEV and guidance for the development of drugs for other diarrhea-causing coronaviruses.

G Protein-Coupled Receptor 37L1 Modulates Epigenetic Changes in Human Renal Proximal Tubule Cells.

Renal luminal sodium transport is essential for physiological blood pressure control, and abnormalities in this process are strongly implicated in the pathogenesis of essential hypertension. Renal G protein-coupled receptors (GPCRs) are critical for the regulation of the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Recently, we showed that GPCR 37L1 (GPR37L1) is expressed on the apical membrane of renal proximal tubules (RPT) and regulates luminal sodium transport and blood pressure by modulating the function of the sodium proton exchanger 3 (NHE3). However, little is known about GPR37L1 intracellular signaling. Here, we show that GPR37L1 is localized to the nuclear membrane, in addition to the plasma membrane in human RPT cells. Furthermore, GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and enhance NHE3 transcription. Overall, we demonstrate the direct role of a nuclear membrane GPCR in the regulation of renal sodium through epigenetic gene regulation.

Downregulation of NHE-3 (SLC9A3) expression by MicroRNAs in intestinal epithelial cells.

Na+/H+ exchanger-3 (NHE-3) is the major apical membrane transporter involved in vectorial Na+ absorption in the intestine. Dysregulation of NHE-3 expression and/or function has been implicated in pathophysiology of diarrhea associated with gut inflammation and infections. Therefore, it is critical to understand the mechanisms involved in the regulation of NHE-3 expression. MicroRNAs (miRNAs) are highly conserved small RNAs that can regulate gene expression at the posttranscriptional level. To date, however, very little is known about the regulation of NHE-3 expression by microRNAs. Therefore, current studies were undertaken to examine the potential miRNA candidates that can regulate the expression of NHE-3 in intestinal epithelial cells. In silico analysis, using different algorithms, predicted several miRNAs that target NHE-3. MicroRNAs with highest context and target score, miR-326, miR-744-5p, and miR-330-5p, were selected for the current study. Human NHE-3 gene 3' untranslated region [3'UTR; 160 base pair (bp)] was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with mimics of miR-326, miR-744-5p, and miR-330-5p into Caco-2, HT-29, and SK-CO15 cells. Cotransfection of NHE-3 3' UTR with miR-326 and -miR-330-5p mimics resulted in a significant decrease in relative luciferase activity. Transfection of miR-326 and -330-5p mimics into SK-CO15 cells significantly decreased the NHE-3 protein expression, with no change in NHE-3 messenger ribonucleic acid (mRNA) levels. Our findings demonstrate a novel mechanism for posttranscriptional regulation of NHE-3 by miR-326 and -330-5p by translational repression. We speculate that miR-326 and -330-5p dependent pathways may be involved in modulating NHE-3 expression under physiological and pathophysiological conditions.

Decreased NHE3 expression in colon cancer is associated with DNA damage, increased inflammation and tumor growth.

Dysregulation of intra- and extracellular pH in cancer contributes to extracellular matrix remodeling, favors cell migration, proliferation, and metastasis. Although the primary attention has been focused on the role of the ubiquitous Na+/H+ exchanger isoform NHE1, the role of NHE3, the predominant apical isoform in colonic surface epithelium in the pathogenesis of colon cancer has not been investigated. Here, we show that NHE3 mRNA expression is significantly reduced in colorectal cancer patients and that low NHE3 expression is associated with poorer survival. Deletion of NHE3 in ApcMin mice evaluated at 15 weeks of age (significant mortality was observed beyond this time) led to lower body weights, increased mucosal inflammation, increased colonic tumor numbers, evidence of enhanced DNA damage in tumor surface epithelium, and to significant alteration in the gut microbiota. In the absence of the inflammatory and microbial pressors, ca. 70% knockdown of NHE3 expression in SK-CO15 cells led to reduced intracellular pH, elevated apical pH, dramatic differences in their transcriptomic profile, increased susceptibility to DNA damage, increased proliferation, decreased apoptosis and reduced adhesion to extracellular matrix proteins. Our findings suggest that loss of NHE3 in the surface epithelium of colonic tumors has profound consequences for cancer progression and behavior.

Intestine-Specific NHE3 Deletion in Adulthood Causes Microbial Dysbiosis.

In the intestine, the Na+/H+ exchanger 3 (NHE3) plays a critical role for Na+ and fluid absorption. NHE3 deficiency predisposes patients to inflammatory bowel disease (IBD). In mice, selective deletion of intestinal NHE3 causes various local and systemic pathologies due to dramatic changes in the intestinal environment, which can influence microbiota colonization. By using metagenome shotgun sequencing, we determined the effect of inducible intestinal epithelial cell-specific deletion of NHE3 (NHE3IEC-KO) in adulthood on the gut microbiome in mice. Compared with control mice, NHE3IEC-KO mice show a significantly different gut microbiome signature, with an unexpected greater diversity. At the phylum level, NHE3IEC-KO mice showed a significant expansion in Proteobacteria and a tendency for lower Firmicutes/Bacteroidetes (F/B) ratio, an indicator of dysbiosis. At the family level, NHE3IEC-KO mice showed significant expansions in Bacteroidaceae, Rikenellaceae, Tannerellaceae, Flavobacteriaceae and Erysipelotrichaceae, but had contractions in Lachnospiraceae, Prevotellaceae and Eubacteriaceae. At the species level, after removing those with lowest occurrence and abundance, we identified 23 species that were significantly expanded (several of which are established pro-inflammatory pathobionts); whereas another 23 species were found to be contracted (some of which are potential anti-inflammatory probiotics) in NHE3IEC-KO mice. These results reveal that intestinal NHE3 deletion creates an intestinal environment favoring the competitive advantage of inflammophilic over anti-inflammatory species, which is commonly featured in conventional NHE3 knockout mice and patients with IBD. In conclusion, our study emphasizes the importance of intestinal NHE3 for gut microbiota homeostasis, and provides a deeper understanding regarding interactions between NHE3, dysbiosis, and IBD.

NHE3 in the thick ascending limb is required for sustained but not acute furosemide-induced urinary acidification.

Na+/H+ exchanger isoform 3 (NHE3) facilitates Na+ reabsorption and H+ secretion by the kidneys. Despite stronger NHE3 abundance in the thick ascending limb (TAL) compared with the S1 and S2 segments of the proximal tubule, the role of NHE3 in the TAL is poorly understood. To investigate the role of NHE3 in the TAL, we generated and phenotyped TAL-specific NHE3 knockout (NHE3TAL-KO) mice. Compared with control mice, NHE3TAL-KO mice did not show significant differences in body weight, blood pH, or plasma Na+, K+, or Cl- levels. Fluid intake trended to be higher and urine osmolality was significantly lower in NHE3TAL-KO mice. Despite a similar glomerular filtration rate, NHE3TAL-KO mice had a greater urinary K+-to-creatinine ratio. One proposed role of NHE3 relates to furosemide-induced urinary acidification. Acute bolus treatment with furosemide under anesthesia did not result in differences in the dose dependence of urinary flow rate, Cl- excretion, or maximal urinary acidification between genotypes; however, in contrast with control mice, urinary pH returned immediately toward baseline levels in NHE3TAL-KO mice. Chronic furosemide treatment reduced urine osmolality similarly in both genotypes but metabolic alkalosis, hypokalemia, and calciuresis were absent in NHE3TAL-KO mice. Compared with vehicle, chronic furosemide treatment resulted in greater Na+-K+-2Cl- abundance regardless of genotype. Na+-phosphate cotransporter 2a abundance was also greater in furosemide-treated control mice compared with vehicle treatment, an effect that was absent in NHE3TAL-KO mice. In summary, NHE3 in the TAL plays a role in the sustained acidification effect of furosemide. Consistent with this, long-term treatment with furosemide did not result in metabolic alkalosis in NHE3TAL-KO mice.NEW & NOTEWORTHY Na+/H+ exchanger isoform 3 (NHE3) is very abundant in the thick ascending limb (TAL) compared with the proximal tubule. Much has been learned about the role of NHE3 in the proximal tubule; however, the function of NHE3 in the TAL remains elusive. A novel mouse model that lacks NHE3 selectively in the TAL not only shows a phenotype under baseline conditions but also identifies that NHE3 is required for sustained but not acute furosemide-induced urinary acidification.

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.

mOrange2, a Genetically Encoded, pH Sensitive Fluorescent Protein, is an Alternative to BCECF-AM to Measure Intracellular pH to Determine NHE3 and DRA Activity.

BACKGROUND/AIMS: NHE3 (Na+/H+ exchanger3) and SLC26A3 (Cl-/HCO3- exchanger, DRA) are the major components of the intestinal neutral NaCl absorptive process and based on the intestinal segment, contribute to HCO3- absorption and HCO3- secretion. NHE3 and DRA are highly regulated by changes in second messengers, cAMP, cGMP and Ca2+. Precise and convenient measurement of exchanger activity is necessary to allow rapid study of physiologic and pharmacologic functions. Some epithelial cells are difficult to load with AM ester dyes and loading may not be uniform. METHODS: The use of a genetically modified fluorescent protein, mOrange2 was explored as an intracellular pH sensor protein to measure exchange activity of NHE3 and DRA. The model used was FRT cells stably expressing NHE3 or DRA with intracellular pH measured by changes of mOrange2 fluorescence intensity. Intracellular pH was monitored using a) Isolated single clones of FRT/mOrange2/HA-NHE3 cells studied in a confocal microscope with time-lapse live cell imaging under basal conditions and when NHE3 was inhibited by exposure to forskolin and stimulated by dexamethasone, b) coverslip grown FRT/mOrange2 cells expressing NHE3 or DRA using a computerized fluorometer with a perfused cuvette with standardization of the mOrange2 absorption and emission signal using K+/Nigericin as an internal standard in each experiment. RESULTS: A similar rate of intracellular alkalization by Na+ addition in cells expressing NHE3 and by Cl- removal in cells expressing DRA was found in mOrange2 expressing cells compared to the same cells loaded with BCECF-AM,both using the same pH calibration with K+/Nigericin. Using mOrange2 as the pH sensor, NHE3 basal activity was quantitated and shown to be inhibited by forskolin and stimulated by dexamethasone, and DRA was oppositely shown to be stimulated by forskolin, responses similar to results found using BCECF-AM. CONCLUSION: This study demonstrates that mOrange2 protein can be an effective alternate to BCECF-AM in measuring intracellular pH (preferred setting Ex520nm, Em 563nm) as affected by NHE3 and DRA activity, with the advantage, compared to AM ester dyes, that genetic expression can provide uniform expression of the pH sensor.

Genetic mutation analysis of 22 patients with congenital absence of vas deferens: a single-center study†.

Congenital absence of the vas deferens (CAVD), a congenital malformation of the male reproductive system, causes obstructive azoospermia and male infertility. Currently, the cystic fibrosis transmembrane conductance regulator (CFTR) has been recognized as the main pathogenic gene in CAVD, with some other genes, such as adhesion G-protein-coupled receptor G2 (ADGRG2), solute carrier family 9 isoform 3 (SLC9A3), sodium channel epithelial 1 subunit beta (SCNN1B), and carbonic anhydrase 12 (CA12), being candidate genes in the pathogenesis of CAVD. However, the frequency and spectrum of these mutations, as well as the pathogenic mechanisms of CAVD, have not been fully investigated. Here, we sequenced all genes with potentially pathogenic mutations using next-generation sequencing and verified all identified variants by Sanger sequencing. Further bioinformatic analysis was performed to predict the pathogenicity of mutations. We described the distribution of the p.V470M, poly-T, and TG-repeat CFTR polymorphisms and identified novel missense mutations in the CFTR and SLC9A3 genes, respectively. Taken together, we identified mutations in the CFTR, ADGRG2, SLC9A3, SCNN1B, and CA12 genes in 22 patients with CAVD, thus broadening the genetic spectrum of Chinese patients with CAVD.

Nedd4-2-dependent Ubiquitination Potentiates the Inhibition of Human NHE3 by Cholera Toxin and Enteropathogenic Escherichia coli.

BACKGROUND & AIMS: Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. METHODS: We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. RESULTS: The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2-hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. CONCLUSIONS: The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans.

The Interplay between Histamine H4 Receptor and the Kidney Function: The Lesson from H4 Receptor Knockout Mice.

Previous studies implicated the histamine H4 receptor in renal pathophysiology. The aim here is to elucidate the role of this receptor on renal function using H4 receptor knockout mice (H4R-/-). Healthy and diabetic H4R-/- mice compared to their C57BL/6J wild-type counterpart for renal function and the expression of crucial tubular proteins. H4R-/- and wild-type mice, matched for ages, showed comparable weight gain curves reaching similar median weight at the end of the study. However, H4R-/- mice displayed a higher basal glycemia. H4R-/- mice showed a lower urine 24 h outflow, and albumin-to-creatinine ratio (ACR) compared to wild-type mice. Consistently, H4R-/- mice presented a higher expression of megalin and a lower basal expression of the sodium-hydrogen exchanger (NHE)3 and aquaporin (AQP)2. According to these basal differences, diabetic H4R-/- mice developed more severe hyperglycemia and a higher 24 h urine volume, but a lower increase in ACR and decrease in urine pH were observed. These events were paralleled by a reduced NHE3 over-expression and megalin loss in diabetic H4R-/- mice. The AQP1 and AQP7 patterns were also different between H4R-/- and wild-type diabetic mice. The collected results highlight the role of the histamine H4 receptor in the control of renal reabsorption processes, particularly albumin uptake.

Reduced activity of intestinal surface Na+/H+ exchanger NHE3 is a key factor for induction of diarrhea after PEDV infection in neonatal piglets.

Porcine epidemic diarrhea virus (PEDV; family Coronaviridae, genus Alphacoronavirus) causes acute diarrhea and vomiting, dehydration, and high mortality in neonatal piglets. Despite extensive research focusing on the pathogenesis of PEDV infection, the molecular pathogenesis of PEDV-induced diarrhea in piglets remains unclear. Na+/H+ exchanger 3 (NHE3), the main exchanger of electroneutral sodium in intestinal epithelial cells, is closely associated with the occurrence of diarrhea. To date, there is no study on whether diarrhea caused by PEDV infection is related to the activity of NHE3. In the present study, it was found that the expression level of cell membrane protein NHE3 significantly decreased after PEDV infection, whereas the total level of protein expression was not significantly changed. The Na+/H+ transport rate and the mRNA abundance of NHE3 decreased; the NHE3 activity decreased gradually with increasing infection time. In vivo, after PEDV infection of newborn piglets, rupture of intestinal villi and interstitial degeneration of intestinal epithelial cells in different intestinal segments were observed by hematoxylin-eosin staining. Immunohistochemical and immunofluorescence methods were used to observe the decreased expression of NHE3 protein on the membrane of intestinal epithelial cells in the jejunum and ileum. Taken together, these data indicate that PEDV infection reduces NHE3 activity in intestinal epithelial cells, hindering Na+ transport and thus causing diarrhea.

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.

DNA methylation in the adipose tissue and whole blood of Agent Orange-exposed Operation Ranch Hand veterans: a pilot study.

BACKGROUND: Between 1962 and 1971, the US Air Force sprayed Agent Orange across Vietnam, exposing many soldiers to this dioxin-containing herbicide. Several negative health outcomes have been linked to Agent Orange exposure, but data is lacking on the effects this chemical has on the genome. Therefore, we sought to characterize the impact of Agent Orange exposure on DNA methylation in the whole blood and adipose tissue of veterans enrolled in the Air Force Health Study (AFHS). METHODS: We received adipose tissue (n = 37) and whole blood (n = 42) from veterans in the AFHS. Study participants were grouped as having low, moderate, or high TCDD body burden based on their previously measured serum levels of dioxin. DNA methylation was assessed using the Illumina 450 K platform. RESULTS: Epigenome-wide analysis indicated that there were no FDR-significantly methylated CpGs in either tissue with TCDD burden. However, 3 CpGs in the adipose tissue (contained within SLC9A3, LYNX1, and TNRC18) were marginally significantly (q < 0.1) hypomethylated, and 1 CpG in whole blood (contained within PTPRN2) was marginally significantly (q < 0.1) hypermethylated with high TCDD burden. Analysis for differentially methylated DNA regions yielded SLC9A3, among other regions in adipose tissue, to be significantly differentially methylated with higher TCDD burden. Comparing whole blood data to a study of dioxin exposed adults from Alabama identified a CpG within the gene SMO that was hypomethylated with dioxin exposure in both studies. CONCLUSION: We found limited evidence of dioxin associated DNA methylation in adipose tissue and whole blood in this pilot study of Vietnam War veterans. Nevertheless, loci in the genes of SLC9A3 in adipose tissue, and PTPRN2 and SMO in whole blood, should be included in future exposure analyses.