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Asthenozoospermia, defined by the absence or reduction of sperm motility, constitutes the most frequent cause of human male infertility. This pathological condition is caused by morphological and/or functional defects of the sperm flagellum, which preclude proper sperm progression. While in the last decade many causal genes were identified for asthenozoospermia associated with severe sperm flagellar defects, the causes of purely functional asthenozoospermia are still poorly defined. We describe here the case of an infertile man, displaying asthenozoospermia without major morphological flagellar anomalies and carrying a homozygous splicing mutation in SLC9C1 (sNHE), which we identified by whole-exome sequencing. SLC9C1 encodes a sperm-specific sodium/proton exchanger, which in mouse regulates pH homeostasis and interacts with the soluble adenylyl cyclase (sAC), a key regulator of the signalling pathways involved in sperm motility and capacitation. We demonstrate by means of RT-PCR, immunodetection and immunofluorescence assays on patient's semen samples that the homozygous splicing mutation (c.2748 + 2 T > C) leads to in-frame exon skipping resulting in a deletion in the cyclic nucleotide-binding domain of the protein. Our work shows that in human, similar to mouse, SLC9C1 is required for sperm motility. Overall, we establish a homozygous truncating mutation in SLC9C1 as a novel cause of human asthenozoospermia and infertility., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Typically, Na + /H + antiporters (NHXs) possess a conserved N-terminus for cation binding and exchange and a hydrophilic C-terminus for regulating the antiporter activity. Plant endosomal-type NHXs play important roles in protein trafficking, as well as K + and vesicle pH homeostasis, however the role of the C-terminal tail remains unclear. Here, the function of MnNHX6, an endosomal-type NHX in mulberry, was investigated using heterologous expression in yeast. Functional and localization analyses of C-terminal truncation and mutations in MnNHX6 revealed that the C-terminal conserved region was responsible for the function and stability of the protein and its hydrophobicity, which is a key domain requirement. Nuclear magnetic resonance spectroscopy provided direct structural evidence and yeast two-hybrid screening indicated that this functional domain was also necessary for interaction with sorting nexin 1. Our findings demonstrate that although the C-terminal tail of MnNHX6 is intrinsically disordered, the C-terminal conserved region may be an important part of the external mouth of this transporter, which controls protein function and stability by serving as an inter-molecular cork with a chain mechanism. These findings improve our understanding of the roles of the C-terminal tail of endosomal-type NHXs in plants and the ion transport mechanism of NHX-like antiporters., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glutamate uptake into synaptic vesicles (SVs) depends on cation/H + exchange activity, which converts the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane at the presynaptic terminals. Thus, the proper recruitment of cation/H + exchanger to SVs is important in determining glutamate quantal size, yet little is known about its localization mechanism. Here, we found that secretory carrier membrane protein 5 (SCAMP5) interacted with the cation/H + exchanger NHE6, and this interaction regulated NHE6 recruitment to glutamatergic presynaptic terminals. Protein-protein interaction analysis with truncated constructs revealed that the 2/3 loop domain of SCAMP5 is directly associated with the C-terminal region of NHE6. The use of optical imaging and electrophysiological recording showed that small hairpin RNA-mediated knockdown (KD) of SCAMP5 or perturbation of SCAMP5/NHE6 interaction markedly inhibited axonal trafficking and the presynaptic localization of NHE6, leading to hyperacidification of SVs and a reduction in the quantal size of glutamate release. Knockout of NHE6 occluded the effect of SCAMP5 KD without causing additional defects. Together, our results reveal that as a key regulator of axonal trafficking and synaptic localization of NHE6, SCAMP5 could adjust presynaptic strength by regulating quantal size at glutamatergic synapses. Since both proteins are autism candidate genes, the reduced quantal size by interrupting their interaction may underscore synaptic dysfunction observed in autism., Competing Interests: The authors declare no competing interest.

Arabidopsis NHX5 and NHX6 are endosomal Na + ,K + /H + antiporters that function in mediating Na + , K + and pH homeostasis. Here, we report that NHX5 and NHX6 mediate Li + homeostasis in Arabidopsis. We found that the nhx5 nhx6 double mutant was defective in growth and had a high pale rate under Li + stress; complementation with either NHX5 or NHX6 restored the growth of the double mutant under LiCl treatments. We further found that CBL3 and CIPK18 collaborate with NHX5 and NHX6 in controlling seedling growth. CBL3 and CIPK18 are involved in the NHX5- and NHX6-mediated response to Li + stress but not to salt or low K + stress. In addition, NHX5 and NHX6 coordinate NHX8, a plasma membrane antiporter, in mediating Li + homeostasis. NHX8 may function differently from NHX5 and NHX6 in mediating Li + homeostasis. NHX8 was not controlled by CBL3 and CIPK18. Overall, CBL3 and CIPK18 are required for the function of NHX5 and NHX6 in mediating Li + homeostasis in Arabidopsis., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The loss of the intestinal Na + /H + exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of Lactobacillus and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine. NEW & NOTEWORTHY Loss of Na + /H + exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.

Salt stress is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple (Malus × domestica). In this study, we demonstrate that a salt-responsive MYB transcription factor (TF), designated as MdMYB63, promotes survival under salt stress. Overexpression of MdMYB63 in apple calli significantly enhanced salt tolerance. Screening of the AP2/ERF family of TFs identified MdERF106 as an interaction partner of MdMYB63. Further analyses showed that the MdMYB63-MdERF106 complex significantly promotes the expression of downstream MdSOS1, thereby improving the Na + expulsion and salt tolerance of apple. These functional analyses of MdMYB63 have provided valuable insights into the regulatory network of salt tolerance, and lay a theoretical foundation for the cultivation of new salt-tolerant apple varieties., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Key Message: Overexpression of K2-NhaD in transgenic cotton resulted in phenotypes with strong salinity and drought tolerance in greenhouse and field experiments, increased expression of stress-related genes, and improved regulation of metabolic pathways, such as the SOS pathway. Drought and salinity are major abiotic stressors which negatively impact cotton yield under field conditions. Here, a plasma membrane Na + /H + antiporter gene, K2-NhaD, was introduced into upland cotton R15 using an Agrobacterium tumefaciens-mediated transformation system. Homozygous transgenic lines K9, K17, and K22 were identified by PCR and glyphosate-resistance. TAIL-PCR confirmed that T-DNA carrying the K2-NhaD gene in transgenic lines K9, K17 and K22 was inserted into chromosome 3, 19 and 12 of the cotton genome, respectively. Overexpression of K2-NhaD in transgenic cotton plants grown in greenhouse conditions and subjected to drought and salinity stress resulted in significantly higher relative water content, chlorophyll, soluble sugar, proline levels, and SOD, CAT, and POD activity, relative to non-transgenic plants. The expression of stress-related genes was significantly upregulated, and this resulted in improved regulation of metabolic pathways, such as the salt overly sensitive pathway. K2-NhaD transgenic plants growing under field conditions displayed strong salinity and drought tolerance, especially at high levels of soil salinity and drought. Seed cotton yields in transgenic line were significantly higher than in wild-type plants. In conclusion, the data indicate that K2-NhaD transgenic lines have great potential for the production of stress-tolerant cotton under field conditions.

Hypertension is a risk factor for premature death and roughly 50% of hypertensive patients are salt-sensitive. The incidence of salt-sensitive hypertension increases with age. However, the mechanisms of salt-sensitive hypertension are not well understood. We had demonstrated decreased renal sodium‑hydrogen exchanger regulatory factor 1 (NHERF1) expression in old salt-resistant F344 rats. Based on those studies we hypothesized that NHERF1 expression is required for the development of some forms of salt-sensitive hypertension. To address this hypothesis, we measured blood pressure in NHERF1 expressing salt-sensitive 4-mo and 24-mo-old male and female Fischer Brown Norway (FBN) rats male and female 18-mo-old NHERF1 knock-out (NHERF1 - / - ) mice and wild-type (WT) littermates on C57BL/6J background after feeding high salt (8% NaCl) diet for 7 days. Our data demonstrate that 8% salt diet increased blood pressure in both male and female 24-mo-old FBN rats but not in 4-mo-old FBN rats and in 18-mo-old male and female WT mice but not in NHERF1 - / - mice. Renal dopamine 1 receptor (D1R) expression was decreased in 24-mo-old rats, compared with 4-mo-old FBN rats. However, sodium chloride cotransporter (NCC) expression increased in 24-mo-old FBN rats. In FBN rats, age had no effect on NaK ATPase α1 and NKCC2 expression. By contrast, high salt diet increased the renal expressions of NKCC2, and NCC in 24-mo-old FBN rats. High salt diet also increased NKCC2 and NCC expression in WT mice but not NHERF1 - / - mice. Our data suggest that renal NHERF1 expression confers salt sensitivity with aging, associated with increased expression of sodium transporters., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose of Review: The current review highlights recent advances in the area of renal tubular phosphate transport and its regulation by fibroblast growth factor 23 (FGF23), a potent regulator of phosphate homeostasis., Recent Findings: Recent studies demonstrate that FGF23 binds to both membrane and soluble form of α-klotho to activate FGF receptor signaling pathways. Parathyroid hormone and FGF23 equivalently decrease sodium-dependent phosphate cotransport but the effect is not additive, suggesting a shared but not synergistic mechanism of action. Crosstalk occurs downstream of parathyroid hormone-receptor and FGF23-receptor signaling and converge at the level of the scaffolding protein, sodium-hydrogen exchanger regulatory factor-1. A novel mechanism for phosphate efflux through the basolateral membrane of renal proximal tubular epithelia via an atypical G-protein coupled receptor, Xenotropic and polytropic retrovirus receptor 1 (XPR1), was recently identified. Conditional deletion of Xpr1 gene in renal proximal tubules in mice leads to hypophosphatemic rickets and Fanconi syndrome establishing an important role for XPR1 in phosphate homeostasis. A novel anti-FGF23 antibody, burosumab, was recently approved to treat X-linked hypophosphatemia, a human disorder of FGF23 excess., Summary: Significant advances in understanding the cellular and molecular aspects of renal tubular phosphate transport and its regulation by FGF23 has led to the discovery of novel therapeutics to treat human disorders of phosphate homeostasis.

Prior studies have demonstrated that the calcium pump, plasma membrane calcium ATPase 2 (PMCA2), mediates calcium transport into milk and prevents mammary epithelial cell death during lactation. PMCA2 also regulates cell proliferation and cell death in breast cancer cells, in part by maintaining the receptor tyrosine kinase ErbB2/HER2 within specialized plasma membrane domains. Furthermore, the regulation of PMCA2 membrane localization and activity in breast cancer cells requires its interaction with the PDZ domain-containing scaffolding molecule sodium-hydrogen exchanger regulatory factor (NHERF) 1. In this study, we asked whether NHERF1 also interacts with PMCA2 in normal mammary epithelial cells during lactation. Our results demonstrate that NHERF1 expression is upregulated during lactation and that it interacts with PMCA2 at the apical membrane of secretory luminal epithelial cells. Similar to PMCA2, NHERF1 expression is rapidly reduced by milk stasis after weaning. Examining lactating NHERF1 knockout (KO) mice showed that NHERF1 contributes to the proper apical location of PMCA2, for proper apical-basal polarity in luminal epithelial cells, and that it participates in the suppression of Stat3 activation and the prevention of premature mammary gland involution. Additionally, we found that PMCA2 also interacts with the closely related scaffolding molecule, NHERF2, at the apical membrane, which likely maintains PMCA2 at the plasma membrane of mammary epithelial cells in lactating NHERF1KO mice. Based on these data, we conclude that, during lactation, NHERF1 is required for the proper expression and apical localization of PMCA2, which, in turn, contributes to preventing the premature activation of Stat3 and the lysosome-mediated cell death pathway that usually occur only early in mammary involution., (Copyright © 2019 Endocrine Society.)

Intracellular Na + /H + antiporters (NHXs) play important roles in plant tolerance to salt stress. However, plant NHXs functioning in salt tolerance and the underlying physiological mechanisms remain poorly understood. In this report, we report the identification and functional characterization of PbrNHX2 isolated from Pyrus betulaefolia. PbrNHX2 expression levels were induced by salt, and dehydration, but was unaffected by cold. PbrNHX2 was localized in the tonoplast. Overexpression of PbrNHX2 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to salt tolerance, whereas down-regulation of PbrNHX2 in Pyrus betulaefolia by virus-induced gene silencing (VIGS) resulted in elevated salt sensitivity. The transgenic lines contained lower levels of Na + , higher levels of K + , and higher K/Na ratio, whereas they were changed in an opposite way when PbrNHX2 was silenced. In addition, the transgenic plants accumulated lower levels of reactive oxygen species compared with wild type, accompanied by higher activities of three antioxidant enzymes. Taken together, the data demonstrate that PbrNHX2 plays a positive role in salt tolerance and that it holds a great potential for engineering salt tolerance in crops., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We now have an increased understanding of the genetics, cell biology, and physiology of electrolyte transport processes in the mammalian intestine, due to the availability of sophisticated methodologies ranging from genome wide association studies to CRISPR-CAS technology, stem cell-derived organoids, 3D microscopy, electron cryomicroscopy, single cell RNA sequencing, transgenic methodologies, and tools to manipulate cellular processes at a molecular level. This knowledge has simultaneously underscored the complexity of biological systems and the interdependence of multiple regulatory systems. In addition to the plethora of mammalian neurohumoral factors and their cross talk, advances in pyrosequencing and metagenomic analyses have highlighted the relevance of the microbiome to intestinal regulation. This article provides an overview of our current understanding of electrolyte transport processes in the small and large intestine, their regulation in health and how dysregulation at multiple levels can result in disease. Intestinal electrolyte transport is a balance of ion secretory and ion absorptive processes, all exquisitely dependent on the basolateral Na + /K + ATPase; when this balance goes awry, it can result in diarrhea or in constipation. The key transporters involved in secretion are the apical membrane Cl - channels and the basolateral Na + -K + -2Cl - cotransporter, NKCC1 and K + channels. Absorption chiefly involves apical membrane Na + /H + exchangers and Cl - /HCO 3 - exchangers in the small intestine and proximal colon and Na + channels in the distal colon. Key examples of our current understanding of infectious, inflammatory, and genetic diarrheal diseases and of constipation are provided. © 2019 American Physiological Society. Compr Physiol 9:947-1023, 2019., (Copyright © 2019 American Physiological Society. All rights reserved.)

Soil salinization is a major abiotic stress condition that affects about half of global agricultural lands. Salinity leads to osmotic shock, ionic imbalance and/or toxicity and build-up of reactive oxygen species. Na⁺/H⁺ antiporters (NHXs) are integral membrane transporters that catalyze the electro-neutral exchange of K⁺/Na⁺ for H⁺ and are implicated in cell expansion, development, pH/ion homeostasis and salt tolerance. Porteresia coarctata is a salt secreting halophytic wild rice that thrives in the coastal-riverine interface. P. coarctata NHX1 (PcNHXI) expression is induced by salinity in P. coarctata roots and shows high sequence identity to Oryza sativa NHX1. PcNHX1 confers hygromycin and Li + sensitivity and Na + tolerance transport in a yeast strain lacking sodium transport systems. Additionally, transgenic PcNHX1 expressing tobacco seedlings (PcNHX1 promoter) show significant growth advantage under increasing concentrations of NaCl and MS salts. Etiolated PcNHX1 seedlings also exhibit significantly elongated hypocotyl lengths in 100 mM NaCl. PcNHX1 expression in transgenic tobacco roots increases under salinity, similar to expression in P. coarctata roots. Under incremental salinity, transgenic lines show reduction in leaf Na + , stem specific accumulation of Na + and K + (unaltered Na + /K + ratios). PcNHX1 transgenic plants also show enhanced chlorophyll content and reduced malondialdehyde (MDA) production in leaves under salinity. The above data suggests that PcNHX1 overexpression (controlled by PcNHX1p) enhances stem specific accumulation of Na + , thereby protecting leaf tissues from salt induced injury., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Na + /H + antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na + /H + antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na + /H + exchanger NHE1, which is an important drug target. Here we resolve the Na + and H + transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.

This communication provides a current overview on the renal protective effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors in diabetics. Following the epoch-making publications, the CANVAS Program and the EMPA-REG OUTCOME trial, numerous literature has discussed the mechanisms by which SGLT2 inhibition exerts its cardio-renal protective effects. Some of them reached agreement, while others did not. This review focuses on the hemodynamic aspect and the remaining potential factors relevant to the renal protection which have not been so much taken up by other review papers. Questions unanswered include factors of uric acid, lipids, erythropoiesis and oxidative stress, salt and sympathetic nerve, and the Na-H exchanger in heart and kidney.

Predicting male fertility is extremely important for artificial insemination and profitable farm management. Conventional semen assessment together with computer-assisted sperm analysis is widely used to predict male fertility under field conditions. However, the clinical validation and sensitivity of these methods remain unclear. Therefore, a new approach is needed to predict male fertility. Here, we investigated the use of a transcriptomic marker (solute carrier family 9, subfamily A, member 3, regulator 1; SLC9A3R1) together with sperm motility parameters and capacitation status to predict fertility/infertility in boars at the commercial level. Our data showed that among motility parameters and the capacitation status, hyperactivation (HYP) differed between high- and low-litter size boars. HYP showed a significant positive correlation (R = 0.468) with boar litter size. Simultaneously, the expression of SLC9A3R1, a gene important in sperm ion channel regulation, was significantly negatively correlated (R = -0.523) with boar litter size. Quality assessment revealed that both HYP and SLC9A3R1 showed considerable sensitivity (71.43 vs. 100%), specificity (100 vs. 71.43%), and overall accuracy (90%) for predicting male fertility. Interestingly, the potential of SLC9A3R1 expression to increase the average piglet number per breeding was higher (0.7 piglets) than that of HYP (0.5 piglets). Thus, measuring SLC9A3R1 expression in spermatozoa may be a more accurate marker for evaluating male fertility/infertility than conventionally used motility parameters and capacitation status., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We studied the role of sodium/proton exchanger 8 (NHE8) in retinal pigment epithelium (RPE) and photoreceptor cells of adult mouse retina by using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Neisseria meningitidis (Nm). Specific single guide RNAs (sgRNAs) were designed to knockdown the Slc9a8 gene, which encodes the NHE8. Nuclease null NmCas9 and sgRNAs were packaged respectively using adeno-associated viral vector (AAV), and delivered into mouse eyes in vivo by subretinal injection on wild-type mice of about four-week-old when mouse retina is fully developed. Eye samples were collected four weeks after injection for phenotype examination. Real-time PCR analysis demonstrated ∼38% reduction of NHE8 transcripts in retinas injected with AAV-knockdown sgRNA and AAV-Cas9. Loss of photoreceptor cells was found in eyes injected with AAV-knockdown sgRNA and AAV-Cas9 under either the human rhodopsin promoter or the minimal chicken β-actin promoter, while normal morphology was observed in control eyes injected with AAV-Cas9 and AAV-control sgRNA; immunostaining data showed degenerating photoreceptor cells and RPE cells in eyes injected with knockdown sgRNA and Cas9 AAVs. We further determined that mutant M120K-NHE8 displayed altered intracellular pH regulation in human RPE and primary mouse RPE cells using genetically encoded pH sensor pHluorin and that primary cultured NHE8 mutant RPE cells showed different pH titration curves. These results indicate that NHE8 plays essential function in both RPE and photoreceptor cells. NHE8 dysfunction either in photoreceptor or RPE is sufficient to cause retinal degeneration in adult mice at any age., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) can be recognized and activated by different pathogen associated molecular patterns (PAMPs), which induce innate immune response and inflammation of the body. Na + /H + exchangers (NHEs) not only play roles in the regulation of cellular pH and cell volume, maintenance of the cavity microenvironment and nutrients absorption, but also are related to cell proliferation, migration and apoptosis. The activity and membrane protein expression of NHEs are inhibited under the inflammation condition. It has been shown that the activation of TLR2 in colon epithelial cells can inhibit the activity of NHE1 through MyD88 independent pathway, which involves the recruitment of Src and the phosphorylation of PI3Ks. Other studies on intestinal macrophage showed long-term LPS stimulation can induce TLR4 activation through MyD88-dependent pathway (TLR4/MyD88/NF-κB) and induce inflammation and degeneration of intracellular NHE1, which leads to NHE1 activity inhibition. But short-term LPS exposure increases the activity and protein expression of NHE1. The activation of TLR5 increases the activity of NHE3. The activity and/or expression of NHE3 in intestinal macrophages in colitis patients and model animals were decreased. In renal tubular epithelial cells, basolateral LPS stimulation inhibits luminal NHE3 activation through TLR4/MyD88-dependent MAPK/ERK signaling pathway. And LPS stimulation on the lumen side activates TLR4/MyD88-dependent PI3K-AKT-mTOR signaling pathway, which results in the inhibition of NHE1 activity in basolateral side, and then affects the NHE3 function of the lumen side.

Diabetes is characterized by increased activity of the sodium-hydrogen exchanger (NHE) in the glomerulus and renal tubules, which contributes importantly to the development of nephropathy. Despite the established role played by the exchanger in experimental studies, it has not been specifically targeted by those seeking to develop novel pharmacological treatments for diabetes. This review demonstrates that many existing drugs that are commonly prescribed to patients with diabetes act on the NHE1 and NHE3 isoforms in the kidney. This action may explain their effects on sodium excretion, albuminuria and the progressive decline of glomerular function in clinical trials; these responses cannot be readily explained by the influence of these drugs on blood glucose. Agents that may affect the kidney in diabetes by virtue of an action on NHE include: (1) insulin and insulin sensitizers; (2) incretin-based agents; (3) sodium-glucose cotransporter 2 inhibitors; (4) antagonists of the renin-angiotensin system (angiotensin converting-enzyme inhibitors, angiotensin receptor blockers and angiotensin receptor neprilysin inhibitors); and (5) inhibitors of aldosterone action and cholesterol synthesis (spironolactone, amiloride and statins). The renal effects of each of these drug classes in patients with type 2 diabetes may be related to a single shared biological mechanism., (© 2017 John Wiley & Sons Ltd.)

The Slc9 family of Na + /H + exchangers (NHEs) plays a critical role in electroneutral exchange of Na + and H + in the mammalian intestine as well as other absorptive and secretory epithelia of digestive organs. These transport proteins contribute to the transepithelial Na + and water absorption, intracellular pH and cellular volume regulation as well as the electrolyte, acid-base, and fluid volume homeostasis at the systemic level. They also influence the function of other membrane transport mechanisms, affect cellular proliferation and apoptosis as well as cell migration, adherence to the extracellular matrix, and tissue repair. Additionally, they modulate the extracellular milieu to facilitate other nutrient absorption and to regulate the intestinal microbial microenvironment. Na + /H + exchange is inhibited in selected gastrointestinal diseases, either by intrinsic factors (e.g., bile acids, inflammatory mediators) or infectious agents and associated bacterial toxins. Disrupted NHE activity may contribute not only to local and systemic electrolyte imbalance but also to the disease severity via multiple mechanisms. In this review, we describe the cation proton antiporter superfamily of Na + /H + exchangers with a particular emphasis on the eight SLC9A isoforms found in the digestive tract, followed by a more integrative description in their roles in each of the digestive organs. We discuss regulatory mechanisms that determine the function of Na + /H + exchangers as pertinent to the digestive tract, their regulation in pathological states of the digestive organs, and reciprocally, the contribution of dysregulated Na + /H + exchange to the disease pathogenesis and progression. © 2018 American Physiological Society. Compr Physiol 8:555-583, 2018., (Copyright © 2018 American Physiological Society. All rights reserved.)

Loss-of-function mutations in human endosomal Na + (K + )/H + exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson syndrome, autism, and attention deficit and hyperactivity disorder. These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not the vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function., (© 2018 Karim and Brett. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na + /H + exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

One of the major goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to fill the knowledge gaps between human genomic information and the corresponding proteomic information. These gaps are due to "missing" proteins (MPs)-predicted proteins with insufficient evidence from mass spectrometry (MS), biochemical, structural, or antibody analyses-that currently account for 2579 of the 19587 predicted human proteins (neXtProt, 2017-01). We address some of the lessons learned from the inconsistent annotations of missing proteins in databases (DB) and demonstrate a systematic proteogenomic approach designed to explore a potential new function of a known protein. To illustrate a cautious and strategic approach for characterization of novel function in vitro and in vivo, we present the case of Na(+)/H(+) exchange regulatory cofactor 1 (NHERF1/SLC9A3R1, located at chromosome 17q25.1; hereafter NHERF1), which was mistakenly labeled as an MP in one DB (Global Proteome Machine Database; GPMDB, 2011-09 release) but was well known in another public DB and in the literature. As a first step, NHERF1 was determined by MS and immunoblotting for its molecular identity. We next investigated the potential new function of NHERF1 by carrying out the quantitative MS profiling of placental trophoblasts (PXD004723) and functional study of cytotrophoblast JEG-3 cells. We found that NHERF1 was associated with trophoblast differentiation and motility. To validate this newly found cellular function of NHERF1, we used the Caenorhabditis elegans mutant of nrfl-1 (a nematode ortholog of NHERF1), which exhibits a protruding vulva (Pvl) and egg-laying-defective phenotype, and performed genetic complementation work. The nrfl-1 mutant was almost fully rescued by the transfection of the recombinant transgenic construct that contained human NHERF1. These results suggest that NHERF1 could have a previously unknown function in pregnancy and in the development of human embryos. Our study outlines a stepwise experimental platform to explore new functions of ambiguously denoted candidate proteins and scrutinizes the mandated DB search for the selection of MPs to study in the future.

Nhe5, a Na + /H + exchanger, is predominantly expressed in brain tissue and is proposed to act as a negative regulator of dendritic spine growth. Up to now, its physiological function in vivo remains unclear. Here we show that Nhe5-deficient mice exhibit markedly enhanced learning and memory in Morris water maze, novel object recognition, and passive avoidance task. Meanwhile, the pre- and post-synaptic components, synaptophysin (Syn) and post-synaptic density 95 (PSD95) expression levels were found increased in hippocampal regions lacking of Nhe5, suggesting a possible alterations in neuronal synaptic structure and function in Nhe5 -/- mice. Further study reveals that Nhe5 deficiency leads to higher Bdnf expression levels, followed by increased phosphorylated TrkB and PLCγ levels, indicating that Bdnf/TrkB signaling is activated due to Nhe5 deficiency. Moreover, the corresponding brain regions of Nhe5 -/- mice display elevated ERK/CaMKII/CREB phosphorylation levels. Taken together, these findings uncover a novel physiological function of Nhe5 in regulating learning and memory, further implying Nhe5 as a potential therapeutic target for improving cognition., (© 2017 Wiley Periodicals, Inc.)

We have shown previously that the durum wheat TdSOS1 excludes Na + and Li + ions outside cells. Moreover, this protein is activated by Arabidopsis kinase SOS2 through phosphorylation. The elimination of both SOS2 phosphorylation sites and the auto-inhibitory domain produces a hyperactive TdSOS1∆972 form, which have a maximal activity independent from the regulatory SOS2/SOS3 complex. We demonstrated that the expression of TdSOS1 enhances salt tolerance of the transgenic Arabidopsis plants. In this study, we analyzed the response to H 2 O 2 -induced oxidative stress of the transgenic Arabidopsis expressing one of the two TdSOS1 forms. Firstly, we showed that the exogenous H 2 O 2 treatment leads to an accumulation of SOS1 transcripts in leaves and roots of the durum wheat and also in the transgenic plants. These transgenic plants showed significant oxidative stress tolerance compared to control plants, especially the plants expressing the hyperactive form. This tolerance was manifested by high proline accumulation and low malonyldialdehyde (MDA), O 2 ˙ - and H 2 O 2 contents. Furthermore, the activities of three essential ROS scavenging enzymes (SOD, CAT, and POD) were higher in the transgenic plants under oxidative stress, as compared to control plants. Taken together, these data suggested that TdSOS1 plays a crucial role in response to oxidative stress.

Objective: To observe the effects of Na(+) /H(+) exchanger 1 (NHE1) inhibitor on intestinal injury of rats with burn sepsis, and to explore the possible mechanism preliminarily. Methods: Ninety SD rats were divided into control group, pure sepsis group, and NHE1 inhibitor group according to the random number table, with 30 rats in each group. Full-thickness scald (hereinafter referred to as burn) model with 20% total body surface area were reproduced on the back of rats in pure sepsis and NHE1 inhibitor groups, and then 50 μL liquid of Pseudomonas aeruginosa ATCC 27853 (2×10(5) colony forming unit/mL) were injected into the center of wounds on the back. Rats in NHE1 inhibitor group were intraperitoneally injected with 0.1 mmol/L NHE1 inhibitor cariporide (0.4 mg/kg) rapidly after the successful establishment of burn sepsis model, while rats in pure sepsis group were injected with the same volume of normal saline. Except for not being made burn wounds nor receiving bacterination, rats in control group were treated the same as those in pure sepsis group. Rats with burn sepsis in each group were laparotomized and injected with 200 mL fluorescein isothiocyanate (FITC)-dextran in the concentration of 0.1 mol/L in terminal ileum at 12 hours post injury, and their left ventricular blood and terminal ileum were collected 30 minutes later. The serum content of FITC-dextran was detected with fluorescence spectrophotometer ( n =10); the morphology of intestinal tissue was observed with HE staining ( n =10); the content of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in serum and intestinal tissue was determined with enzyme-linked immunosorbent assay ( n =20); the activity of myeloperoxidase (MPO) in serum and intestinal tissue was detected with colorimetric method ( n =20); the protein expression of nuclear factor-kappa B-p65 (NF-κB-p65) and phosphorylation levels of mitogen-activated protein kinase (MAPK) signal pathway related proteins p38MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase 1/2 (JNK1/2) were determined by Western blotting ( n =4). The same samples of rats in control group were collected for related detection at the same time point as above. Data were processed with one-way analysis of variance and SNK test. Results: (1) The serum content of FITC-dextran of rats in pure sepsis group was significantly higher than that in control group ( P <0.01), while the serum content of FITC-dextran of rats in NHE1 inhibitor group was significantly lower than that in pure sepsis group ( P <0.01). Compared with that in control group, infiltration of a large number of inflammatory cells, ulcer and necrosis of intestinal mucosa of rats in pure sepsis group were observed. The injury condition of intestine of rats in NHE1 inhibitor group was better than that in pure sepsis group. (2) The serum content of IL-6, TNF-α, and MPO of rats in pure sepsis group was (387±42) and (164.7±10.1) ng/mL, and (7.5±1.5) U/mL, respectively, significantly higher than that in control group [(75±17) and (13.1±6.5) ng/mL, and (2.3±0.7) U/mL, respectively, with P values below 0.01]. The serum content of IL-6, TNF-α, and MPO of rats in NHE1 inhibitor group was (176±37) and (64.9±9.3) ng/mL, and (5.9±0.8) U/mL, respectively, which was significantly lower than that in pure sepsis group (with P values below 0.01). (3) The content of IL-6, TNF-α, and MPO in intestinal tissue of rats in pure sepsis group was (190±13) and (172.8±29.7) ng/mL, and (8.7±1.5) U/mL, respectively, significantly higher than that in control group [respectively (20±3) and (11.9±2.3) ng/mL, and (2.9±0.3) U/mL, with P values below 0.01]. The content of IL-6, TNF-α, and MPO of intestinal tissue of rats in NHE1 inhibitor group was (35±6) and (45.2±6.1) ng/mL, and (5.3±0.6) U/mL, respectively, significantly lower than that in pure sepsis group (with P values below 0.01). (4) The protein expression of NF-κB-p65 and phosphorylation levels of p38MAPK and ERK1/2 in intestinal tissue of rats in pure sepsis group were significantly higher than those in control group (with P values below 0.01); the protein expression of NF-κB-p65 and the phosphorylation level of p38MAPK in intestinal tissue of rats in NHE1 inhibitor group were significantly lower than those in pure sepsis group (with P values below 0.01); phosphorylation levels of JNK1/2 in intestinal tissue of rats in the three groups were similar (with P values above 0.05). Conclusions: The inhibition of NHE1 can significantly alleviate the intestinal injury, and the mechanisms may be attributed to the regulation of NF-κB and p38MAPK signal pathway, resulting in inhibition of the inflammatory response of intestinal tract.

Key Points: Intracellular pH regulation is vital to neurons as nerve activity produces large and rapid acid loads in presynaptic terminals. Rapid clearance of acid loads is necessary to maintain control of neurotransmission, but neuronal acid clearance mechanisms remain poorly understood. Glutamate is loaded into synaptic vesicles via the vesicular glutamate transporter (VGLUT), a mechanism conserved across phyla, and this study reports a previously unknown role for VGLUT as an acid-extruding protein when deposited in the plasmamembrane during exocytosis. The finding was made in Drosophila (fruit fly) larval motor neurons through a combined pharamacological and genetic dissection of presynaptic pH homeostatic mechanisms. A dual role for VGLUT serves to integrate neuronal activity and pH regulation in presynaptic nerve terminals., Abstract: Neuronal activity can result in transient acidification of presynaptic terminals, and such shifts in cytosolic pH (pH cyto ) probably influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals, we hypothesized that the same activity might drive acid efflux mechanisms to maintain pH cyto homeostasis. To better understand the integration of neuronal activity and pH cyto regulation we investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically encoded pH indicator, named 'pHerry', in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity-induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H + /Na + exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid-extruding protein when deposited on the plasmamembrane., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

The Na(+)/H(+)-exchanger NHE1 and the monocarboxylate transporters MCT1 and MCT4 are crucial for intracellular pH regulation, particularly under active metabolism. NHE1, a reversible antiporter, uses the energy provided by the Na(+) gradient to expel H(+) ions generated in the cytosol. The reversible H(+)/lactate(-) symporters MCT1 and 4 cotransport lactate and proton, leading to the net extrusion of lactic acid in glycolytic tumors. In the first two sections of this article we review important features and remaining questions on the structure, biochemical function and cellular roles of these transporters. We then use a fully-coupled mathematical model to simulate their relative contribution to pH regulation in response to lactate production, as it occurs in highly hypoxic and glycolytic tumor cells. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Membrane-bound pyrophosphatases (mPPases) hydrolyze pyrophosphate (PPi) to transport H(+), Na(+) or both and help organisms to cope with stress conditions, such as high salinity or limiting nutrients. Recent elucidation of mPPase structure and identification of subfamilies that have fully or partially switched from Na(+) to H(+) pumping have established mPPases as versatile models for studying the principles governing the mechanism, specificity and evolution of cation transporters. In the present study, we constructed an accurate phylogenetic map of the interface of Na(+)-transporting PPases (Na(+)-PPases) and Na(+)- and H(+)-transporting PPases (Na(+),H(+)-PPases), which guided our experimental exploration of the variations in PPi hydrolysis and ion transport activities during evolution. Surprisingly, we identified two mPPase lineages that independently acquired physiologically significant Na(+) and H(+) cotransport function. Na(+),H(+)-PPases of the first lineage transport H(+) over an extended [Na(+)] range, but progressively lose H(+) transport efficiency at high [Na(+)]. In contrast, H(+)-transport by Na(+),H(+)-PPases of the second lineage is not inhibited by up to 100 mM Na(+) With the identification of Na(+),H(+)-PPase subtypes, the mPPases protein superfamily appears as a continuum, ranging from monospecific Na(+) transporters to transporters with tunable levels of Na(+) and H(+) cotransport and further to monospecific H(+) transporters. Our results lend credence to the concept that Na(+) and H(+) are transported by similar mechanisms, allowing the relative efficiencies of Na(+) and H(+) transport to be modulated by minor changes in protein structure during the course of adaptation to a changing environment., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The NHE1 isoform of the mammalian Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells by removing one intracellular proton in exchange for one extracellular sodium. Deletion of the NHE1 gene (SLC9A1) affects the growth and motor ability of mice and humans but mutations and polymorphisms of the gene are only beginning to be characterized. NHE1 has a cytosolic C-terminal regulatory tail of approximately 315 amino acids and a 500 amino acid membrane domain. We examined the functional effects of three human stop codon mutations at amino acids 321, 449 and 735 in comparison with a mutant that had a shortened tail region (543 stop codon). The short mutants, 321, 449 and 543 stop codon mutant proteins, lost NHE1 activity and expression, and did not target to the plasma membrane. Protein for these short mutants was more rapidly degraded than the wild type and 735 ending proteins. The 735 terminating mutant, with the membrane domain and much of the cytosolic tail, had reduced protein expression and activity. The results demonstrate that early stop codon polymorphisms have significant and deleterious effects on the activity of the SLC9A1 protein product. The 735-NHE1 mutant, without the last 80 amino acids, had more minor defects. Surprisingly, retention of a proximal 43 amino acids adjacent to the membrane domain did little to maintain NHE1 expression, targeting and activity., Competing Interests: The authors have declared that no competing interests exist.

Microglia play important roles in extracellular matrix remodeling, tumor invasion, angiogenesis, and suppression of adaptive immunity in glioma. Na(+)/H(+) exchanger isoform 1 (NHE1) regulates microglial activation and migration. However, little is known about the roles of NHE1 in intratumoral microglial activation and microglia-glioma interactions. Our study revealed up-regulation of NHE1 protein expression in both glioma cells and tumor-associated Iba1(+) microglia in glioma xenografts and glioblastoma multiforme microarrays. Moreover, we observed positive correlation of NHE1 expression with Iba1 intensity in microglia/macrophages. Glioma cells, via conditioned medium or non-contact glioma-microglia co-cultures, concurrently upregulated microglial expression of NHE1 protein and other microglial activation markers (iNOS, arginase-1, TGF-β, IL-6, IL-10 and the matrix metalloproteinases MT1-MMP and MMP9). Interestingly, glioma-stimulated microglia reciprocally enhanced glioma proliferation and migration. Most importantly, inhibition of microglial NHE1 activity via small interfering RNA (siRNA) knockdown or the potent NHE1-specific inhibitor HOE642 significantly attenuated microglial activation and abolished microglia-stimulated glioma migration and proliferation. Taken together, our findings provide the first evidence that NHE1 function plays an important role in glioma-microglia interactions, enhancing glioma proliferation and invasion by stimulating microglial release of soluble factors. NHE1 upregulation is a novel marker of the glioma-associated microglial activation phenotype. Inhibition of NHE1 represents a novel glioma therapeutic strategy by targeting tumor-induced microglial activation., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

It is well recognized that the renin-angiotensin system (RAS) exists not only as circulating, paracrine (cell to cell), but also intracrine (intracellular) system. In the kidney, however, it is difficult to dissect the respective contributions of circulating RAS versus intrarenal RAS to the physiological regulation of proximal tubular Na(+) reabsorption and hypertension. Here, we review recent studies to provide an update in this research field with a focus on the proximal tubular RAS in angiotensin II (ANG II)-induced hypertension. Careful analysis of available evidence supports the hypothesis that both local synthesis or formation and AT1 (AT1a) receptor- and/or megalin-mediated uptake of angiotensinogen (AGT), ANG I and ANG II contribute to high levels of ANG II in the proximal tubules of the kidney. Under physiological conditions, nearly all major components of the RAS including AGT, prorenin, renin, ANG I, and ANG II would be filtered by the glomerulus and taken up by the proximal tubules. In ANG II-dependent hypertension, the expression of AGT, prorenin, and (pro)renin receptors, and angiotensin-converting enzyme (ACE) is upregulated rather than downregulated in the kidney. Furthermore, hypertension damages the glomerular filtration barrier, which augments the filtration of circulating AGT, prorenin, renin, ANG I, and ANG II and their uptake in the proximal tubules. Together, increased local ANG II formation and augmented uptake of circulating ANG II in the proximal tubules, via activation of AT1 (AT1a) receptors and Na(+)/H(+) exchanger 3, may provide a powerful feedforward mechanism for promoting Na(+) retention and the development of ANG II-induced hypertension.

Christianson syndrome (CS) is an X-linked neurodevelopmental and neurological disorder characterized in males by core symptoms that include non-verbal status, intellectual disability, epilepsy, truncal ataxia, postnatal microcephaly and hyperkinesis. CS is caused by mutations in the SLC9A6 gene, which encodes a multipass transmembrane sodium (potassium)-hydrogen exchanger 6 (NHE6) protein, functional in early recycling endosomes. The extent and variability of the CS phenotype in female heterozygotes, who presumably express the wild-type and mutant SLC9A6 alleles mosaically as a result of X-chromosome inactivation (XCI), have not yet been systematically characterized. Slc9a6 knockout mice (Slc9a6 KO) were generated by insertion of the bacterial lacZ/β-galactosidase (β-Gal) reporter into exon 6 of the X-linked gene. Mutant Slc9a6 KO male mice have been shown to develop late endosomal/lysosomal dysfunction associated with glycolipid accumulation in selected neuronal populations and patterned degeneration of Purkinje cells (PCs). In heterozygous female Slc9a6 KO mice, β-Gal serves as a transcriptional/XCI reporter and thus facilitates testing of effects of mosaic expression of the mutant allele on penetrance of the abnormal phenotype. Using β-Gal, we demonstrated mosaic expression of the mutant Slc9a6 allele and mosaically distributed lysosomal glycolipid accumulation and PC pathology in the brains of heterozygous Slc9a6 KO female mice. At the behavioral level, we showed that heterozygous female mice suffer from visuospatial memory and motor coordination deficits similar to but less severe than those observed in X-chromosome hemizygous mutant males. Our studies in heterozygous Slc9a6 KO female mice provide important clues for understanding the likely phenotypic range of Christianson syndrome among females heterozygous for SLC9A6 mutations and might improve diagnostic practice and genetic counseling by helping to characterize this presumably underappreciated patient/carrier group., (© 2016. Published by The Company of Biologists Ltd.)

Background: Na(+)/H(+) exchanger-1 (NHE-1) is involved in pH regulation and is up-regulated in different malignancies. Activation of NHE-1 is one way for allowing cells to avoid intracellular acidification and protect them against apoptosis. Inhibitors of NHE-1 are able to decrease intracellular pH and induce apoptosis. Some statins can also act by partial inhibition of NHE-1. This review presents progress in understanding the mechanisms of action of these inhibitors, connections with certain genetic mutations and acquired treatment resistance, as well as new patents on them., Methods: A MEDLINE search for original and review articles using key terms, Na(+)/H(+) exchanger, leukemia, cariporide, and amiloride. Recent patents with NHE-1 inhibitors published by United States Patent and Trademark Office are also presented., Results and Conclusions: Sorafenib is used for the treatment of acute myeloid leukemia patients carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation. 5-(N, N-hexamethylene)-amiloride can increase the suppression of FLT3 signaling by sorafenib. NHE-1 inhibitors are able to increase the sensitivity of chronic myeloid leukemia cells to tyrosine kinase inhibitors, including through the inhibition of P-glycoprotein. NHE-1 inhibitors are promising adjuvant drugs for overcoming acquired resistance to treatment in various malignant hemopathies.

Background: The presence of intracellular pH (pHi ) regulators, including Na(+) -H(+) exchanger (NHE), Na(+) -HCO3- co-transporter (NBC), Cl(-) /OH(-) exchanger (CHE), and Cl(-) /HCO3- exchanger (AE), have been confirmed in many mammalian cells. Alcohol consumption is associated with increased risk of cardiovascular disorder. The aims of the study were to identify the possible transmembrane pHi regulators and to explore the effects of ethanol (EtOH) (10 to 300 mM) on the resting pHi and pHi regulators in human aorta smooth muscle cells (HASMCs)., Methods: HASMCs were obtained from patients undergoing heart transplant. The pHi was measured by microspectrofluorimetry with the pH-sensitive dye, BCECF-AM., Results: The following results are obtained. (i) In cultured HASMCs, the resting pHi was 7.19 ± 0.04 and 7.13 ± 0.02 for HEPES- and CO2 /HCO3--buffered solution, respectively. (ii) Two different Na(+) -dependent acid-equivalent extruders, including NHE and Na(+) -coupled HCO3- transporter, functionally coexisted. (iii) Two different Cl(-) -dependent acid loaders (CHE and AE) were functionally identified. (iv) EtOH induced a biphasic, concentration-dependent change in resting pHi (+0.25 pH unit at 100 mM but only +0.05 pH unit at 300 mM) in bicarbonate-buffered solution, while caused a concentration-dependent decrease in resting pHi (-0.06 pH unit at 300 mM) in HEPES-buffered solution. (v) The effect of EtOH on NHE activity was also biphasic: increase of 40% at lower concentration of 10 mM, followed by decrease of 30% at higher concentration of 300 mM. (vi) The increase in Na(+) -coupled HCO3- transporter activity by EtOH was concentration dependent. (vii) The effect of EtOH on CHE and AE activities was both biphasic: increase of ~25% at 30 mM, followed by decrease of 10 to 25% at 100 mM, and finally increase of 15 to 20% at 300 mM., Conclusions: This study demonstrated that 2 acid extruders and 2 acid loaders coexisted functionally in HASMCs and that EtOH induced a biphasic, concentration-dependent change in resting pHi by altering the activity of the 2 acid extruders, NHE and Na(+) -coupled HCO3- transporter, and the 2 acid loaders, CHE and AE., (Copyright © 2015 by the Research Society on Alcoholism.)

Loss of mineralocorticoid receptor signaling selectively in cardiomyocytes can ameliorate cardiac fibrotic and inflammatory responses caused by excess mineralocorticoids. The aim of this study was to characterize the role of cardiomyocyte mineralocorticoid receptor signaling in ischemia-reperfusion injury and recovery and to identify a role of mineralocorticoid receptor modulation of cardiac function. Wild-type and cardiomyocyte mineralocorticoid receptor knockout mice (8 weeks) were uninephrectomized and maintained on (1) high salt (0.9% NaCl, 0.4% KCl) or (2) high salt plus deoxycorticosterone pellet (0.3 mg/d, 0.9% NaCl, 0.4% KCl). After 8 weeks of treatment, hearts were isolated and subjected to 20 minutes of global ischemia plus 45 minutes of reperfusion. Mineralocorticoid excess increased peak contracture during ischemia regardless of genotype. Recovery of left ventricular developed pressure and rates of contraction and relaxation post ischemia-reperfusion were greater in knockout versus wild-type hearts. The incidence of arrhythmic activity during early reperfusion was significantly higher in wild-type than in knockout hearts. Levels of autophosphorylated Ca(2+)/calmodulin protein kinase II (Thr287) were elevated in hearts from wild-type versus knockout mice and associated with increased sodium hydrogen exchanger-1 expression. These findings demonstrate that cardiomyocyte-specific mineralocorticoid receptor-dependent signaling contributes to electromechanical vulnerability in acute ischemia-reperfusion via a mechanism involving Ca(2+)/calmodulin protein kinase II activation in association with upstream alteration in expression regulation of the sodium hydrogen exchanger-1., (© 2015 American Heart Association, Inc.)

Potassium (K(+) ) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. CHX14 expression was induced by elevated K(+) and histochemical analysis of CHX14 promoter::GUS transgenic plants indicated that CHX14 was expressed in xylem parenchyma of root and shoot vascular tissues of seedlings. CHX14 knockout (chx14) and CHX14 overexpression seedlings displayed different growth phenotypes during K(+) stress as compared with wild-type seedlings. Roots of mutant seedlings displayed higher K(+) uptake rates than wild-type roots. CHX14 expression in yeast cells deficient in K(+) uptake renders the mutant cells more sensitive to deficiencies of K(+) in the medium. CHX14 mediates K(+) efflux in yeast cells loaded with high K(+) . Uptake experiments using (86) Rb(+) as a tracer for K(+) with both yeast and plant mutants demonstrated that CHX14 expression in yeast and in planta mediated low-affinity K(+) efflux. Functional green fluorescent protein (GFP)-tagged versions of CHX14 were localized to both the yeast and plant plasma membranes. Taken together, we suggest that CHX14 is a plasma membrane K(+) efflux transporter involved in K(+) homeostasis and K(+) recirculation., (© 2015 John Wiley & Sons Ltd.)

Diarrhea associated with ulcerative colitis (UC) occurs primarily as a result of reduced Na(+) absorption. Although colonic Na(+) absorption is mediated by both epithelial Na(+) channels (ENaC) and Na-H exchangers (NHE), inhibition of NHE-mediated Na(+) absorption is the primary cause of diarrhea in UC. As there are conflicting observations reported on NHE expression in human UC, the present study was initiated to identify whether NHE isoforms (NHE2 and NHE3) expression is altered and how Na(+) absorption is regulated in DSS-induced inflammation in rat colon, a model that has been used to study UC. Western blot analyses indicate that neither NHE2 nor NHE3 expression is altered in apical membranes of inflamed colon. Na(+) fluxes measured in vitro under voltage clamp conditions in controls demonstrate that both HCO3 (-)-dependent and butyrate-dependent Na(+) absorption are inhibited by S3226 (NHE3-inhibitor), but not by HOE694 (NHE2-inhibitor) in normal animals. In contrast, in DSS-induced inflammation, butyrate-, but not HCO3 (-)-dependent Na(+) absorption is present and is inhibited by HOE694, but not by S3226. These observations indicate that in normal colon NHE3 mediates both HCO3 (-)-dependent and butyrate-dependent Na(+) absorption, whereas DSS-induced inflammation activates NHE2, which mediates butyrate-dependent (but not HCO3 (-)-dependent) Na(+) absorption. In in vivo loop studies HCO3 (-)-Ringer and butyrate-Ringer exhibit similar rates of water absorption in normal rats, whereas in DSS-induced inflammation luminal butyrate-Ringer reversed water secretion observed with HCO3 (-)-Ringer to fluid absorption. Lumen butyrate-Ringer incubation activated NHE3-mediated Na(+) absorption in DSS-induced colitis. These observations suggest that the butyrate activation of NHE2 would be a potential target to control UC-associated diarrhea., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: The intercellular adhesion molecule 1 (ICAM-1) is induced in mouse liver after bile duct ligation (BDL) and plays a key role in neutrophil-mediated liver injury in BDL mice. ICAM-1 has been shown to interact with cytoskeletal ezrin-radixin-moesin (ERM) proteins that also interact with the PDZ protein, Na(+) /H(+) exchanger regulatory factor 1 (NHERF-1/EBP50). In NHERF-1(-/-) mice, ERM proteins are significantly reduced in brush-border membranes from kidney and small intestine. ERM knockdown reduces ICAM-1 expression in response to tumor necrosis factor alpha. Here we show that NHERF-1 assembles ERM proteins, ICAM-1 and F-actin into a macromolecule complex that is increased in mouse liver after BDL. Compared to wild-type (WT) mice, both sham-operated and BDL NHERF-1(-/-) mice have lower levels of activated ERM and ICAM-1 protein in the liver accompanied by significantly reduced hepatic neutrophil accumulation, serum alanine aminotransferase, and attenuated liver injury after BDL. However, total bile acid concentrations in serum and liver of sham and BDL NHERF-1(-/-) mice were not significantly different from WT controls, although hepatic tetrahydroxylated bile acids and Cyp3a11 messenger RNA levels were higher in NHERF-1(-/-) BDL mice., Conclusion: NHERF-1 participates in the inflammatory response that is associated with BDL-induced liver injury. Deletion of NHERF-1 in mice leads to disruption of the formation of ICAM-1/ERM/NHERF-1 complex and reduction of hepatic ERM proteins and ICAM-1, molecules that are up-regulated and are essential for neutrophil-mediated liver injury in cholestasis. Further study of the role of NHERF-1 in the inflammatory response in cholestasis and other forms of liver injury should lead to discovery of new therapeutic targets in hepatic inflammatory diseases., (© 2015 by the American Association for the Study of Liver Diseases.)

The cation/proton antiporter (CPA) family includes the well-known sodium/proton exchanger (NHE; SLC9A) family of Na(+)/H(+) exchangers, and the more recently discovered and less well understood CPA2s (SLC9B), found widely in living organisms. In Drosophila, as in humans, they are represented by two genes, Nha1 (Slc9b1) and Nha2 (Slc9b2), which are enriched and functionally significant in renal tubules. The importance of their role in organismal survival has not been investigated in animals, however. Here we show that single RNAi knockdowns of either Nha1 or Nha2 reduce survival and in combination are lethal. Knockdown of either gene alone results in up-regulation of the other, suggesting functional complementation of the two genes. Under salt stress, knockdown of either gene decreases survival, demonstrating a key role for the CPA2 family in ion homeostasis. This is specific to Na(+) stress; survival on K(+) intoxication is not affected by sodium/hydrogen antiporter (NHA) knockdown. A direct functional assay in Xenopus oocytes shows that Nha2 acts as a Na(+)/H(+) exchanger. In contrast, Nha1 expressed in Xenopus oocytes shows strong Cl(-) conductance and acts as a H(+)-Cl(-) cotransporter. The activity of Nha1 is inhibited by chloride-binding competitors 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and 4,4'-dibenzamido-2,2'-stilbenedisulphonate. Salt stress induces a massive up-regulation of NHA gene expression not in the major osmoregulatory tissues of the alimentary canal, but in the crop, cuticle, and associated tissues. Thus, it is necessary to revise the classical view of the coordination of different tissues in the coordination of the response to osmoregulatory stress.

Intermittent hypoxia (IH) occurs frequently in patients with obstructive sleep apnoea and can cause ventricular dysfunction. However, whether myocardial inflammation and sodium-hydrogen exchanger-1 (NHE-1) expression play an important role in IH-induced ventricular dysfunction remains unclear. This study aimed to investigate whether short-term exercise provides a protective effect on IH-induced left ventricular (LV) function impairment. Male Sprague-Dawley rats were randomly assigned to 4 groups: control (CON), IH, exercise (EXE) or IH interspersed with EXE (IHEXE). IH rats were exposed to repetitive hypoxia/reoxygenation cycles (2%-6% O₂ for 2-5 s per 75 s, followed by 21% O₂ for 6 h/day) during the light phase for 12 consecutive days. EXE rats were habituated to treadmill running for 5 days, permitted 2 days of rest, and followed by 5 exercise bouts (30 m/min for 60 min on a 2% grade) on consecutive days during the dark phase. IHEXE rats were exposed to IH during the light phase interspersed with exercise programs during the dark phase on the same day. Cardiac function was quantified by echocardiographic evaluation. Myocardial levels of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and NHE-1 were determined. IH rats showed LV dysfunction characterized by lower LV fractional shortening (LVFS%) and LV ejection fraction (LVEF%). LV dysfunction was associated with higher myocardial levels of TNF-α, IL-6 and NHE-1 mRNA and protein. These changes were not observed in IHEXE rats (P > 0.05 for all). EXE rats showed lower levels of NHE-1 protein than CON rats (P < 0.05). However, the levels of LVFS%, LVEF%, TNF-α and IL-6 protein and NHE-1 mRNA did not differ between EXE and CON rats (P > 0.05 for all). These data indicated that exercise may provide a protective effect on IH-induced LV dysfunction by attenuating IH-induced myocardial NHE-1 hyperactivity.

A common complication of type 1 diabetes mellitus is diabetic ketoacidosis (DKA), a state of severe insulin deficiency. A potentially harmful consequence of DKA therapy in children is cerebral edema (DKA-CE); however, the mechanisms of therapy-induced DKA-CE are unknown. Our aims were to identify the DKA treatment factors and membrane mechanisms that might contribute specifically to brain cell swelling. To this end, DKA was induced in juvenile mice with the administration of the pancreatic toxins streptozocin and alloxan. Brain slices were prepared and exposed to DKA-like conditions in vitro. Cell volume changes were imaged in response to simulated DKA therapy. Our experiments showed that cell swelling was elicited with isolated DKA treatment components, including alkalinization, insulin/alkalinization, and rapid reductions in osmolality. Methyl-isobutyl-amiloride, a nonselective inhibitor of sodium-hydrogen exchangers (NHEs), reduced cell swelling in brain slices elicited with simulated DKA therapy (in vitro) and decreased brain water content in juvenile DKA mice administered insulin and rehydration therapy (in vivo). Specific pharmacological inhibition of the NHE1 isoform with cariporide also inhibited cell swelling, but only in the presence of the anion transport (AT) inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid. DKA did not alter brain NHE1 isoform expression, suggesting that the cell swelling attributed to the NHE1 was activity dependent. In conclusion, our data raise the possibility that brain cell swelling can be elicited by DKA treatment factors and that it is mediated by NHEs and/or coactivation of NHE1 and AT., (Copyright © 2015 the American Physiological Society.)

The euryhaline pupfish, Cyprinodon variegatus variegatus (Cvv), can successfully osmoregulate in ≥2 mM Na(+) and a freshwater population (Cyprinodon variegatus hubbsi; Cvh) osmoregulates at ≥0.1mM Na(+). We previously demonstrated that Cvv relies on an apical NKCC and NHE in the gill for Na(+) uptake in high (7mM) and intermediate (2 mM) Na(+) concentrations, while Cvh relies only on NHE for Na(+) uptake. This study investigated whether differential NHE isoform use explains differences in Na(+) uptake kinetics between these two populations. We further studied whether Cvh uses a NHE-Rh metabolon or carbonic anhydrase (CA) to overcome thermodynamic challenges of NHE function in dilute freshwater. Transfer to more dilute freshwater resulted in upregulation of nhe-2 (Cvv only) and nhe-3 (Cvv and Cvh). Relative expression of nhe-3 compared to nhe-2 was 2-fold higher in Cvv, but 200-fold higher in Cvh suggesting that nhe-3 expression is an important freshwater adaptation for Cvh. Simultaneous measurement of Na(+) and Tamm flux under various conditions provided no support for a NHE-Rh metabolon in either population. Carbonic anhydrase activity in Cvv was comparable in 7 and 2 mM Na(+) acclimated fish. In Cvh, CA activity increased by 75% in 0.1 mM Na(+) acclimated fish compared to 7 mM Na(+) fish. Ethoxzolamide had variable effects, stimulating and reducing Na(+) uptake in Cvv acclimated to 7 and 2 mM Na(+), while reducing Na(+) uptake in 7 and 0.1mM Na(+) acclimated Cvh. This suggests that CA plays important, but different roles in regulating Na(+) uptake in Cvv and Cvh., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Studies using pharmacological and genetic approaches have shown that increased activity/expression of the Na+/H+ exchanger isoform 1 (NHE1) play a critical role in the pathogenesis of cardiac hypertrophy. Despite the importance of NHE1 in cardiac hypertrophy, severe cerebrovascular side effects were associated with the use of NHE1 inhibitors when administered to patients with myocardial infarctions. p90 ribosomal S6 Kinase (RSK), a downstream regulator of the mitogen-activated protein kinase pathway, has also been implicated in cardiac hypertrophy. We hypothesized that RSK plays a role in the NHE1 induced cardiomyocyte hypertrophic response. Infection of H9c2 cardiomyoblasts with the active form of the NHE1 adenovirus induced hypertrophy and was associated with an increase in the phosphorylation of RSK (P<0.05). Parameters of hypertrophy such as cell area, protein content and atrial natriuretic mRNA expression were significantly reduced in H9c2 cardiomyoblasts infected with active NHE1 in the presence of dominant negative RSK (DN-RSK) (P<0.05). These results confirm that NHE1 lies upstream of RSK. Increased phosphorylation and activation of GATA4 at Ser261 was correlated with increased RSK phosphorylation. This increase was reversed upon inhibition of RSK or NHE1. These findings demonstrate for the first time that the NHE1 mediated hypertrophy is accounted for by increased activation and phosphorylation of RSK, which subsequently increased the phosphorylation of GATA4; eventually activating fetal gene transcriptional machinery.

A new N-ethyl-N-nitrosourea (ENU)-induced mouse recessive mutation, identified by fundus examination of the eye, develops depigmented patches, indicating retinal disorder. Histology data show aberrant retinal pigment epithelium (RPE) and late-onset photoreceptor cell loss in the mutant retina. Chromosomal mapping and DNA sequencing reveal a point mutation (T to A) of the Slc9a8 gene, resulting in mutant sodium/proton exchanger 8 (NHE8)-M120K protein. The lysine substitution decreases the probability of forming the 3(rd) transmembrane helix, which impairs the pore structure of the Na(+)/H(+) exchanger. Various RPE defects, including mislocalization of the apical marker ezrin, and disrupted apical microvilli and basal infoldings are observed in mutant mice. We have further generated NHE8 knockout mice and confirmed similar phenotypes, including abnormal RPE cells and late-onset photoreceptor cell loss. Both in vivo and in vitro data indicate that NHE8 co-localizes with ER, Golgi and intracellular vesicles in RPE cells. Thus, NHE8 function is necessary for the survival of photoreceptor cells and NHE8 is important for RPE cell polarity and function. Dysfunctional RPE may ultimately lead to photoreceptor cell death in the NHE8 mutants. Further studies will be needed to elucidate whether or not NHE8 regulates pH homeostasis in the protein secretory pathways of RPE.

Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB‑231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1‑EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.

The sodium-hydrogen exchanger isoform-1 [NHE1] is a ubiquitously expressed plasma membrane protein that plays a central role in intracellular pH and cell volume homeostasis by catalyzing an electroneutral exchange of extracellular sodium and intracellular hydrogen. Outside of this important physiological function, the NHE1 cytosolic tail domain acts as a molecular scaffold regulating cell survival and actin cytoskeleton organization through NHE1-dependent signaling proteins. NHE1 plays main roles in response to physiological stress conditions which in addition to cell shrinkage and acidification, include hypoxia and mechanical stimuli, such as cell stretch. NHE1-mediated modulation of programmed cell death results from the exchanger-mediated changes in pHi, cell volume, and/or [Na+]I; and, it has recently become known that regulation of cellular signaling pathways are involved as well. This review focuses on NHE1 functions and regulations. We describe evidence showing how these structural actions integrate with ion translocation in regulating renal tubule epithelial cell survival., (© 2015 S. Karger AG, Basel.)

HCO3(-) reabsorption in the renal tubules plays a critically important role in maintaining the global acid-base balance. Loss of HCO3(-) causes metabolic acidosis. Proximal renal tubule is the major site for HCO3(-) reabsorption, accounting for more than 80% of total HCO3(-) reabsorption along the nephron. Over the past more than half centuries, tremendous progresses have been made on understanding the molecular mechanisms underlying the HCO3(-) reabsorption in proximal tubules. The transepithelial movement of HCO3(-) involves the coordinated operation of machineries on both the apical and the basolateral membranes of the epithelial cells. On the apical domain, Na(+)-H(+) exchanger NHE3 and the vacuolar H(+)-ATPase are two major pathways mediating the apical uptake of HCO3(-)-related species. Taken together, NHE3 and H(+)-ATPase are responsible for about 80% of HCO3(-) reabsorption in the proximal tubule. The remaining 20% is likely mediated by pathways yet to be characterized. On the basolateral membrane, NBCe1 represents the only major known pathway mediating the extrusion of HCO3(-) coupled with Na(+) into the interstitial space. In the present article, we provide a historical view about the studies on the mechanisms of HCO3(-) reabsorption since 1940s. Moreover, we summarize the latest progresses emerging over the past decade in the physiological as well as pathological roles of acid-base transporters underlying the HCO3(-) reabsorption in proximal tubules.