NHE Activity Research Articles

Evaluating the Role of the Na+‐H+ Exchanger Isoform 1 (NHE1) in Lysophosphatidic Acid and Urokinase‐type Plasminogen Activator Stimulation of Ovarian Cancer Cells

Lysophosphatidic Acid (LPA) is a bioactive lipid which stimulates an increase in cell proliferation and migration in a range of normal and cancer lines in part through the activation of both the RhoA/Rock and Ras/ErK signaling pathways. Plasma levels of LPA are elevated in ovarian cancer patients even in the early stages of the disease where it is believed to participate in pathogenesis. In patients with more advanced stages of ovarian cancer, the higher the levels of LPA found in the blood and ascites is a negative prognostic indicator. In ovarian cancer, LPA enhances cell proliferation and promotes cell survival by preventing apoptosis and anoikis. LPA also decreases the sensitivity of the ovarian cancer cells to cisplatin and other chemotherapy drugs. While LPA signaling can directly enhance ovarian cancer progression, it can also act by stimulating the production and release of urokinase‐type plasminogen activator (uPA). uPA is a serine protease that binds to a plasma membrane receptor stimulating activation of both the RhoA/Rock and Ras/ErK signaling pathways. Studies in non‐small cell lung cancer cells have shown that LPA and uPA activate the Na+‐H+ Exchanger Isoform 1 (NHE1) leading to an increase in cell proliferation and migration. NHE1 is an 815 amino acid transmembrane protein that exchanges 1 extracellular Na+ for 1 intracellular H+ leading to the pH inversion common to cancer cells in a developing tumor. Activation of NHE1 is one of the initial steps in the development of the transformed phenotype of cancer cells. NHE1 can be phosphorylated and activated by Rock and kinases in the Ras/Erk signaling pathway. Recently, increases in NHE1 expression was demonstrated to play an important role in cancer progression in ovarian cancer patients. In this project, we specifically explore the relationship between LPA and uPA signaling to NHE1 in the regulation of cell proliferation, stress fiber formation, and cell migration in three ovarian cancer cell lines, CAOV‐3, SKOV‐3, and OVCAR‐3.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyperglycemia‐Induced Alteration of Brain Microvascular Endothelial Intracellular Ca Response to Ischemic Factors: Role of TRPV4 Channels

Cerebral edema is exacerbated in hyperglycemic ischemic stroke through poorly understood mechanisms. We showed previously that hypoxia and other factors present during the early hours of ischemic stroke stimulate blood‐brain barrier (BBB) Na‐K‐Cl cotransport (NKCC1) and Na/H exchange (NHE1) activities, leading to increased secretion of Na, Cl and water from blood into brain across a still intact BBB and consequent edema formation. More recently we found that BBB endothelial cells exposed to high glucose (6 hr to 7 d) exhibit increased abundance of NKCC and NHE and greater stimulation of the transporter activities by ischemic factors compared to cells maintained in normoglycemic conditions. Further, we found that the more robust edema formation observed in hyperglycemic rats is significantly attenuated by bumetanide and HOE‐642, inhibitors of NKCC1 and NHE1 activities. The present study was conducted to investigate signaling events underlying these effects of hyperglycemia on BBB Na transporters and ischemia‐induced cerebral edema with the overall goal of identifying additional therapeutic targets for diabetic ischemic stroke. Compelling evidence suggests that intracellular Ca (Cai) signaling mechanisms are likely to be involved. Exposure to high glucose is known to alter Cai dynamics in several cell types, including non‐brain endothelial cells. In addition, stimulation of NKCC and/or NHE activity by increases in Cai has been demonstrated in a variety of cell types. In recent studies we found that exposing BBB endothelial cells to high glucose for 48 hours resulted in elevated resting levels of Cai and significantly increased the abundance of the Transient Receptor Potential V4 (TRPV4) channel. The present study was conducted to further evaluate the effects of hyperglycemia on BBB endothelial cell Cai and to investigate whether TRPV4 plays a role in hyperglycemia‐induced alteration of Cai dynamics in the cells. Bovine cerebral microvascular endothelial cells (CMEC) were exposed for 24 hr to hyperglycemic (HG, 30mM glucose), normoglycemic (NG, 5mM glucose) or osmotic control media (5mM glucose + 12.5 mM NaCl) then loaded with 5μM Cal‐520‐AM and imaged after exposure (1 hr) to normoxia or hypoxia (7% O2). Calcium levels were normalized to maximal non‐saturated calcium signal following the addition of 8μM ionomycin. We found that HG CMEC exhibited a 4‐fold increase in [Cai] when exposed to hypoxia compared to a 2‐fold in NG CMEC. This effect was abolished in the presence Ca‐free media or the TRPV4 inhibitor HC067047 (1μM). Western blot evaluation of CMEC lysates following exposure to HG media (6 to 48 hr) revealed significant increases in TRPV4 abundance after 24 and 48 hr but no effect of osmotic control medium (NG media with added 12.5 mM NaCl). Using conventional perforated patch‐clamp methods and a 1‐second voltage ramp (−100 mV to +100 mV) protocol, we also found that the TRPV4 agonist GSK1016790A activated an outwardly rectifying current in the cells that was blocked by the antagonist (HC067047; 1μM), indicating the presence of TRPV4 activity in the cells. Our findings indicate that hyperglycemia exposures cause more robust hypoxia‐induced increases in BBB endothelial cell [Cai] in a TRPV4‐dependent manner.Support or Funding InformationSupported by NINDS and the American Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression and Regulation of CHP and NHE in Cancer

The hypoxic and serum starved environment of metastasizing cancer cells requires a mechanism to overcome the low intracellular pH induced by the Warburg effect. The sodium hydrogen exchanger isoform 1 (NHE1) is a highly regulated, transmembrane protein that modulates pH of extra and intra cellular environment. One set of proteins which bind and regulate NHE1 include calcineurin homologous proteins 1 and 2 (CHP1&2). CHP1 is necessary for basal NHE1 transport while CHP2, increases the exchange activity and Vmax of NHE1. Both proteins bind to nearly the same NHE1 residues however these interactions are not clearly known. To understand the role that all three proteins may play in regulating cellular function, tissue expressional distribution was conducted on tumor and non‐diseased human tissues. Analysis of RNA sequence from Expression Atlas database revealed that different from published record, CHP2 is expressed within a number non‐diseased tissue including but not limited to: spinal cord, urinary bladder, vagina, tibial nerve, lower leg skin. Analysis of RNA sequence databases of cancerous tissue showed significantly higher expressed CHP2 within a number of, but not all cancer tissues including lung, cervical, and esophogeal cancers. Surprisingly prostate and colorectal adenocarcinoma express CHP2 with a 3–10 fold decrease in expression compared to non‐diseased tissue. To further analyse the expression of CHP2 and NHE1 in lung cancer cells, we performed qPCR on human tissue samples. Cq values displayed a significantly higher expression of CHP2 within squamous cell carcinoma, but not adenocarcinoma, in comparison to adjacent healthy tissue. RNAseq, Expression Atlas, analysis of CHP2 within non‐small cell lung cancer also demonstrates this 15‐fold increased expression for squamous cell carcinoma. This signals an integral role of CHP2 for the survival of cancer cells, and hints at the subsequent impact upon the regulation of NHE1 to allow cells to survive nascent tumour acidification. To examine this possibility, we measured the impact of pHi in several cancer cell lines treated with an NHE1 specific inhibitor or after NHE1 or CHP2 knockdown. This work shows that CHP2 expression is required for cells to maintain sustainable pHi for survival. The impact of NHE1 in hypoxic conditions several tumor cell lines. Thus, the increase in expression and change in transport kinetics demonstrates the effect of CHP2 upon proton transport for NHE1, recovering pHi and increasing cell survival.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inducible intestinal epithelial cell‐specific NHE3 knockout causes diarrhea and more alkaline luminal content

The Na+/H+ exchanger isoform 3 (NHE3) has an important role in intestinal and renal Na+ uptake, acid‐base regulation, and composition of the gut microbiota. Reduced intestinal NHE3 activity and/or expression were found in congenital Na+ diarrhea and inflammatory bowel disease. Previously, no viable intestinal epithelial cell‐specific NHE3 knockout model has been generated; whole body NHE3 knockout mice show 70% mortality and non‐inducible intestinal epithelial cell‐specific NHE3 knockout mice show 100% postnatal mortality. Therefore, we generated a tamoxifen‐inducible intestinal epithelial‐specific NHE3 knockout mouse (NHE3IEC‐Ko; see abstract #1519). Two weeks after tamoxifen administration, NHE3IEC‐Ko mice show ~2‐fold increase in intestinal permeability, 4 hours after oral gavage of FITC‐Dextran, compared to control mice (P<0.05). The small intestine, cecum, and colon were markedly dilated in NHE3IEC‐Ko versus control mice, which was associated with a more than 2‐fold increase in total intestinal wet weight (4.9±0.5 vs 2.2±0.1 g, P<0.05, n=4 and 8, respectively) and ~1.5‐fold increase in fecal water content (55±2 vs 81±4 %, P<0.05). Small intestinal and colonic flush pH were 0.5 pH and 0.8 pH units more alkaline, respectively, in NHE3IEC‐Ko compared to control mice (both P<0.05). Small intestinal length was not different between genotypes (36±3 vs 39±1 cm, NS), but colon length was increased in NHE3IEC‐Ko compared to control mice (8.8±0.3 vs 7.1±0.2 cm, P<0.05). Although there was not an increase in intestinal length, the proximal small intestine of NHE3IEC‐Ko mice had longer villi (493±4 vs 392±19 μm, P<0.05) but similar crypt depths compared to control mice (89±4 vs 79±3 μm, NS). No differences in villus length (159±11 vs 139±7 μm, NS) or crypt depth (58±2 vs 58±2 μm, NS) were observed in the distal small intestine between genotypes. In conclusion, this novel intestinal epithelial cell‐specific NHE3 knockout model is a useful tool for studying inflammatory bowel disease, and the role of intestinal pH on the microbiome as well as intestinal proliferation and apoptosis.Support or Funding InformationNIH 1R01DK110621, APS STRIDE Undergraduate Summer Research Fellowship 1R25HL115473‐01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Empagliflozin effects on ischemic contracture and I/R injury in isolated mouse hearts perfused with or without insulin

IntroductionThe kidney‐targeted Empagliflozin (EMPA) has shown large unexpected cardiovascular benefits, which cannot be explained by general cardiovascular risk factors. We recently reported that EMPA has sodium hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes. Classic NHE inhibitors such as Cariporide (CARI) delay ischemic contracture onset and reduce ischemia reperfusion (I/R) injury in the heart. Here we examined whether EMPA delays contracture onset and reduces I/R injury. Both normal‐ and low‐glycogen hearts were used, because the rate of glycogen depletion during ischemia affects contracture onset and IR injury.Methods/ResultsIsolated mouse hearts were perfused with and without 50 mU/L insulin, to create normal‐ and low‐glycogen hearts respectively, and subjected to 25 min I and 120 min R. 1μM EMPA, 10μM cariporide (CARI; positive control) or vehicle was administered prior to ischemia until 10 minutes post‐reperfusion. In normal‐glycogen hearts, EMPA and CARI did not change ischemic contracture (in seconds, vehicle 1056±56, EMPA 900±44, CARI 880±84) and IR injury (infarct size (%): vehicle 59±6, Empa 62±10, Cari 68±4), however in low‐glycogen hearts, both EMPA and CARI delayed ischemic contracture onset (vehicle 459±25, EMPA 559±22, CARI 588±31). Higher EMPA concentration further delayed contracture onset. Only CARI reduced I/R injury (from 51±6 to 34±5). The presence of insulin (same concentration used) did not alter NHE activity, nor the ability of CARI or EMPA to inhibit NHE, in isolated cardiomyocytes. The increased IR injury in normal‐glycogen hearts was associated with increased end‐ischemic lactate and glucose‐6‐phosphate accumulation, and consequently decreased mitochondrial hexokinase ll.ConclusionEMPA does not protect against I/R injury in isolated hearts, yet EMPA delayed ischemic contracture onset in low‐glycogen hearts, suggesting a direct cardiac, ATP preserving, effect of EMPA during ischemia. Cardiac glycogen content interferes with the effects of NHE inhibitors on I/R injury in isolated hearts. These data indicate that empagliflozin and cariporide may inhibit NHE in different manners.Support or Funding InformationNone.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proline‐Rich Tyrosine Kinase Phosphorylation's Effect On the Na+/H+ Exchanger Isoform 1

The Na+‐H+ exchanger isoform 1 (NHE1) is a 12‐pass transmembrane protein that functions by exchanging one extracellular sodium ion for one intracellular proton. The transmembrane domain of NHE1 is involved in ion exchange, and the cytoplasmic tail is the regulatory domain which includes a series of phosphorylation sites as well as lipid and proteins binding sites that alter transport activity. NHE1 is a central driver of the hallmarks of cancer through its involvement in the regulation of cellular proliferation, migration, and invasion. While the exact number of phosphorylation sites on the cytoplasmic domain of NHE1 is still unclear, many studies have implicated 22 phosphorylation sites and 12 different kinases. One of the kinases believed to directly or indirectly lead to the phosphorylation of NHE1 at S602 and S605 is proline rich tyrosine kinase 2 (PyK2). To evaluate the role of PyK2 in NHE1 regulation we prepared three cell lines derived from Chinese hamster lung fibroblast lacking NHE1 expression (PS120 cells). These cell lines are PSN (PS120 cells expressing human NHE1), PSN S602A/S605A (PSN cells with serine to alanine mutations of the reported PyK2 phosphorylation sites) and PSN S602D/S605D (PSN cells with serine to aspartic acid mutations of the reported PyK2 phosphorylation sites to mimic phosphorylation). Some studies have found that PyK2 works inversely on NHE1 and causes inhibition upon phosphorylation, while other studies have shown PyK2 to enhance NHE1 activity. Here we evaluate the impact of PyK2 inhibition and mutation of the PyK2 phosphorylation sites on proliferation and migration. Western blot analysis is used to evaluate changes in Pyk2 expression and phosphorylation over time under low and normal serum conditions. Cell proliferation and cell migration are both evaluated. Our results suggest that serum stimulation increases Pyk2 phosphorylation and enhances both cell proliferation and cell migration. Inhibition of PyK2 by PF43I396 or the mutation of the Pyk2 phosphorylation sites on NHE1 decreases proliferation and migration rates in serum stimulated cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Palmitoylation Regulates the Functions of NHE1

The sodium hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane protein that regulates intercellular pH (pHi) and interacts with proteins involved in cell migration and adhesion. Within the extended cytosolic tail of NHE1, there are multiple sites of post‐translational modification including phosphorylation and lipidation. Our understanding of impact of reversible palmitoylation on membrane transporters is growing. 2‐bromopalmitate (2BP) irreversibly inhibits the enzyme, palmitoyl acyltransferase, that transfer palmitate to cysteines. We found NHE1 is reversibly palmitoylated and here, demonstrate the impact of the lipidation on NHE1 function. Lung fibroblasts expressing NHE1 (PSN) showed minimal decrease (8.5 ± 2.5% SEM) in cell viability after treatment with 50 μM 2BP after 72 h. Palmitoylation plays a critical role in cell adhesion and motility. Cells that were incubated in 15 μM 2BP for 18 hrs displayed a significant decrease cell adhesion in low serum conditions and cells treated with 2BP did not fully migrate into vacated space after 24 hrs in a wounding assay. Impact on cytoskeletal structure by 2BP treatment was also investigated. Lysophosphatidic acid (LPA) increased actin stress fiber formation 40% over control cells. This LPA‐induced stress fiber formation was inhibited more than two‐fold in the presence of 2BP. This is important as LPA‐induced stress fiber formation requires NHE1; therefore, palmitoylation is required for NHE1‐dependent actin stress fiber formation. To determine the effect of palmitoylation on NHE1 transport activity, PSN cells were treated with 2BP and the pHi was measured. Addition of increasing concentrations of 2BP inhibited NHE1 transport in cells with and without serum, but the effect was enhanced in low serum conditions. Interestingly, LPA stimulation of NHE1 activity was inhibited by 60%, but not to baseline. This indicates that in addition to cell adherence, motility, and cytoskeletal formation, palmitoylation is critical for NHE1 transport.Support or Funding InformationThis research was partially funded by the Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating the Potential for Na+‐H+ Exchanger Isoform 1 (NHE1) Inhibitors as Adjuvant Therapies in the Treatment of Ovarian Cancer

Each year in the United States over 200,000 women are diagnosed with ovarian cancer leading to more than 130,000 deaths. A major challenge in ovarian cancer treatment is the development of drug resistance to the therapeutic agents. Increases in the expression and activity Na+‐H+ Exchanger Isoform 1 (NHE1) was recently demonstrated to regulate cell proliferation in ovarian cancer patients. NHE1 is an 815 amino acid transmembrane transport protein that regulates intracellular pH. We hypothesize that combining common ovarian cancer therapeutic agents along with compounds that directly or indirectly inhibit NHE1 will enhance the ability of the therapeutic agents to slow ovarian cancer cell proliferation and migration. Three ovarian adenocarcinoma cell lines; CAOV‐3, SKOV‐3, and OVCAR‐3 are used in this research. Using a proliferation assay, we assessed the impact of different ovarian cancer therapeutics (Rucaparib, Olaparib, Paclitaxel and Cisplatin) in these three cell lines in the presence and absence of inhibitors that decrease NHE1 activity directly or indirectly. Our previous work demonstrated that the NHE1 inhibitor cariporide and a number of protein kinase inhibitors decreased cell proliferation rates in all three cell types. Treatment with Cisplatin reduced cell proliferation most significantly in the CAOV‐3 line. Paclitaxel and Cisplatin each had significant and comparable impacts on the SKOV‐3 and OVCAR‐3 cell lines. Cell migration was evaluated using electric cell‐substrate impedance sensing (ECIS). In the SKOV‐3 and CAOV‐3 cells, inhibition with cariporide and the Rsk inhibitor BID1870 each dramatically reduced the rate of cell migration. The research presented here supports the role of NHE1 in cell proliferation, and for the first time implicates NHE1 in the regulation of cell migration in ovarian cancer cells. Data will be presented that evaluated the potential for the inhibition of NHE1 as an adjuvant therapy in ovarian cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SLC9 Gene Family: Function, Expression, and Regulation.

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.

Predicted effect of circadian clock modulation of NHE3 of a proximal tubule cell on sodium transport.

Major renal functions such as renal blood flow, glomerular filtration rate, and urinary excretion are known to exhibit circadian oscillations. However, the underlying mechanisms that govern these variations have yet to be fully elucidated. To better understand the impact of the circadian clock on renal solute and water transport, we have developed a computational model of the renal circadian clock and coupled that model to an epithelial transport model of the proximal convoluted cell of the rat kidney. The activity of the Na+-H+ exchanger 3 (NHE3) is assumed to be regulated by changes in transcription of the NHE3 mRNA due to regulation by circadian clock proteins. The model predicts the rhythmic oscillations in NHE3 activity, which gives rise to significant daily fluctuations in Na+ and water transport of the proximal tubule cell. Additionally, the model predicts that 1) mutation in period 2 (Per2) or cryptochrome 1 (Cry1) preserves the circadian rhythm and modestly raises Na+ reabsorption; 2) mutation in Bmal1 or CLOCK eliminates the circadian rhythm and modestly lowers Na+ reabsorption; 3) mutation in Rev-Erb or ROR-related orphan receptor (Ror) has minimal impact on the circadian oscillations. The model represents the first step in building a tool set aimed at increasing our understanding of how the molecular clock affects renal ion transport and renal function, which likely has important implications for kidney disease.

Effect of cariporide on ram sperm pH regulation and motility: possible role of NHE1.

Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium-hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent Km for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.

Na+/H+ exchanger isoform 1-induced osteopontin expression facilitates cardiac hypertrophy through p90 ribosomal S6 kinase.

Cardiovascular diseases are the leading cause of death worldwide. One in three cases of heart failure is due to dilated cardiomyopathy. The Na+/H+ exchanger isoform 1 (NHE1), a multifunctional protein and the key pH regulator in the heart, has been demonstrated to be increased in this condition. We have previously demonstrated that elevated NHE1 activity induced cardiac hypertrophy in vivo. Furthermore, the overexpression of active NHE1 elicited modulation of gene expression in cardiomyocytes including an upregulation of myocardial osteopontin (OPN) expression. To determine the role of OPN in inducing NHE1-mediated cardiomyocyte hypertrophy, double transgenic mice expressing active NHE1 and OPN knockout were generated and assessed by echocardiography and the cardiac phenotype. Our studies showed that hearts expressing active NHE1 exhibited cardiac remodeling indicated by increased systolic and diastolic left ventricular internal diameter and increased ventricular volume. Moreover, these hearts demonstrated impaired function with decreased fractional shortening and ejection fraction. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA was upregulated, and there was an increase in heart cell cross-sectional area confirming the cardiac hypertrophic effect. Moreover, NHE1 transgenic mice also showed increased collagen deposition, upregulation of CD44 and phosphorylation of p90 ribosomal s6 kinase (RSK), effects that were regressed in OPN knockout mice. In conclusion, we developed an interesting comparative model of active NHE1 transgenic mouse lines which express a dilated hypertrophic phenotype expressing CD44 and phosphorylated RSK, effects which were regressed in absence of OPN.

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters.

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

Identifying the localization and exploring a functional role for Gprc5c in the kidney

The investigation of orphan GPCRs (GPRs) has the potential to uncover novel insights into whole animal physiology. In this study, our goal was to determine the renal localization of Gprc5c, a receptor that we previously reported to be highly expressed in murine whole kidney, and to examine physiologic parameters in Gprc5c knockout (KO) mice to gain insight into function. Gprc5c localized to the apical membrane of renal proximal tubules (PTs) in mice, rats, and humans. With the comparison of Gprc5c wild-type (WT) and KO mice, we found that Gprc5c KO mice have altered acid-base homeostasis. Specifically, Gprc5c KO mice have lower blood pH and higher urine pH compared with WT mice, with a reduced level of titratable acids in their urine. In an in vitro GPCR internalization assay, we observed that Gprc5c internalization (an index of activation) was triggered by alkaline extracellular pH. Furthermore, with the use of an in vitro BCECF assay, we observed that Gprc5c increases Na+/H+ exchanger 3 (NHE3) activity at alkaline pH. We also find that the NHE3 activity is reduced in Gprc5c KO mice by 2 photon imaging in seminaphthorhodafluors (SNARF)-4F-loaded kidney sections. NHE3 is a primary contributor to apical transport of H+ in the renal PT. Together, these data imply that Gprc5c modulates the renal contribution to systemic pH homeostasis, at least in part, by taking part in the regulation of NHE3.-Rajkumar, P., Cha, B., Yin, J., Arend, L. J., Păunescu, T. G., Hirabayashi, Y., Donowitz, M., Pluznick, J. L. Identifying the localization and exploring a functional role for Gprc5c in the kidney.

The Angiotensin II Type 1 Receptor Antagonist Losartan Affects NHE1-Dependent Melanoma Cell Behavior

Background/Aims: The peptide hormone angiotensin II (ATII) plays a prominent role in regulating vasoconstriction and blood pressure. Its primary target is the angiotensin II receptor type 1 (AT1), the stimulation of which induces an increase in cytosolic [Ca2+] and calmodulin activation. Ca2+-bound (activated) calmodulin stimulates the activity of the Na+/ H+ exchanger isoform 1 (NHE1); and increased NHE1 activity is known to promote melanoma cell motility. The competitive AT1 receptor inhibitor losartan is often used to lower blood pressure in hypertensive patients. Since AT1 mediates ATII-stimulated NHE1 activity, we set out to investigate whether ATII and losartan have an impact on NHE1-dependent behavior of human melanoma (MV3) cells. Methods: ATII receptor expression was verified by PCR, F-actin was visualized using fluorescently labeled phalloidin, and cytosolic [Ca2+] and pH were determined ratiometrically using Fura-2 and BCECF, respectively. MV3 cell behavior was analyzed using migration, adhesion, invasion and proliferation assays. Results: MV3 cells express both AT1 and the angiotensin II receptor type 2 (AT2). Stimulation of MV3 cells with ATII increased NHE1 activity which could be counteracted by both losartan and the Ca2+/ calmodulin inhibitor ophiobolin-A. ATII stimulation induced a decrease in MV3 cell migration and a more spherical cell morphology accompanied by an increase in the density of F-actin. Independently of the presence of ATII, both NHE1 and migratory activity were reduced when AT1 was blocked by losartan. On the other hand, losartan clearly increased cell adhesion to, and the invasion of, a collagen type I substrate. The AT2 inhibitor PD123319 did not affect NHE1 activity, proliferation and migration, but increased adhesion and invasion. Conclusion: Losartan inhibits NHE1 activity and the migration of human melanoma cells. At the same time, losartan promotes MV3 cell adhesion and invasion. The therapeutic use of AT1 antagonists (sartans) in hypertensive cancer patients should therefore be given critical consideration.

No Change in Bicarbonate Transport but Tight-Junction Formation Is Delayed by Fluoride in a Novel Ameloblast Model

We have recently developed a novel in vitro model using HAT-7 rat ameloblast cells to functionally study epithelial ion transport during amelogenesis. Our present aims were to identify key transporters of bicarbonate in HAT-7 cells and also to examine the effects of fluoride exposure on vectorial bicarbonate transport, cell viability, and the development of transepithelial resistance. To obtain monolayers, the HAT-7 cells were cultured on Transwell permeable filters. We monitored transepithelial resistance (TER) as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using the fluorescent indicator BCECF. Activities of ion transporters were tested by withdrawal of various ions from the bathing medium, by using transporter specific inhibitors, and by activation of transporters with forskolin and ATP. Cell survival was estimated by alamarBlue assay. Changes in gene expression were monitored by qPCR. We identified the activity of several ion transporters, NBCe1, NHE1, NKCC1, and AE2, which are involved in intracellular pH regulation and vectorial bicarbonate and chloride transport. Bicarbonate secretion by HAT-7 cells was not affected by acute fluoride exposure over a wide range of concentrations. However, tight-junction formation was inhibited by 1 mM fluoride, a concentration which did not substantially reduce cell viability, suggesting an effect of fluoride on paracellular permeability and tight-junction formation. Cell viability was only reduced by prolonged exposure to fluoride concentrations greater than 1 mM. In conclusion, cultured HAT-7 cells are functionally polarized and are able to transport bicarbonate ions from the basolateral to the apical fluid spaces. Exposure to 1 mM fluoride has little effect on bicarbonate secretion or cell viability but delays tight-junction formation, suggesting a novel mechanism that may contribute to dental fluorosis.

The mechanism of action of sodium–glucose co‐transporter 2 inhibitors is similar to carbonic anhydrase inhibitors

Butler et al.,1 in their review about the potential role of sodium–glucose co-transporter-2 inhibitors (SGLT2i) in the treatment of heart failure (HF), do not mention the carbonic anhydrase (CA) inhibitors, whose mechanism of action has remarkable similarities with SGLT2i. Although there are no studies in humans,2 it is established that around 30% of the filtered Na+ is reabsorbed through the Na+/H+ isoform 3 (NHe3) exchanger, which is located in the proximal convoluted tubule (PCT), whereas up to 15% of tubular Na+ is reabsorbed through SGLT2 in situations of hyperglycaemia. The SGLT2i inhibit both the SGLT2 and the NHe3 exchangers that share the microdomain with SGLT2,2 and they have a natriuretic effect independent of the glycaemic level.3 On the other hand, NHe3 is activated in HF and is involved in the states of resistance to diuretics.4 Carbonic anhydrase is an enzyme located on the luminal membrane of PCT cells that accelerates the formation of H2CO3 and its subsequent conversion into H2O and CO2 using H+ and HCO3−, which favours the mobilization of NHe3 substrates and increases the tubular reabsorption of Na+. CA inhibitors, such as acetazolamide, act to inhibit the CA, slowing down the H2CO3 circuit and decreasing NHe3 activity, leading to natriuresis.5 The SGLT2i and CA inhibitors indirectly inhibit NHe3, cause mild to moderate natriuresis and bicarbonaturia, and activate the juxtaglomerular system by decreasing hyperfiltration and proteinuria, secondary to increased Na+ and Cl− reabsorption by macula densa (MD). This reabsorption would activate the juxtaglomerular system allowing the local secretion of adenosine that in turn stimulates the afferent arteriole vasoconstriction, which leads to a decrease in hyperfiltration. This haemodynamic fact does not occur with loop diuretics, thiazides, or K+-sparing diuretics. The loop diuretics block the carrier Na+/K+/2Cl− in the MD, and the others act beyond the ascending loop of Henle and MD, so these drugs would have no proteinuric or nephroprotective effect.5 There are differences in the natriuretic effect between SGLT2i and CA inhibitors. CA inhibitors lose their diuretic effect in the long term due to compensatory mechanisms of distal reabsorption.5 However, the SGLT2i maintain their long-term diuretic effect. Thus, currently, the clinical use of acetazolamide in HF is restricted to patients with diuretic resistance. In contrast, the diuretic effect of SGLT2i is maintained over time, probably due to the glucosuric effect6 that modifies renin levels.1 Despite these differences in their long-term temporal effect considering the similarities in the mechanism of action of SGLT2i and CA inhibitors, and considering the benefit demonstrated by SGLT2i, it would be interesting to develop specific clinical trials with CA inhibitors in patients with diabetes and HF (https://clinicaltrials.gov/ct2/show/NCT01973335).

Evidence for a role of PDZ domain-containing proteins to mediate hypophosphatemia in calcium stone formers.

Hypophosphatemia (HYP) is common among calcium stone formers (SFs) and in rare cases is associated with mutations in sodium-phosphate cotransporters or in Na+/H+ exchanger regulatory factor 1 (NHERF1), but the majority of cases are unexplained. We hypothesized that reduced sodium-phosphate cotransporter activity mediated via NHERF1 or a similar PDZ domain-containing protein, causes HYP. If so, other transport activities controlled by NHERF1, such as NHE3 and URAT1, might be reduced in HYP. To test this idea, we analyzed two large but separate sets of 24-h urine samples and paired serums of 2700 SFs from the University of Chicago and 11 073 SFs from Litholink, a national laboratory. Patients were divided into quintiles based on serum phosphate. Males were more common in the lowest phosphate tiles in both datasets. Phosphate excretion did not vary across the quintiles, excluding diet as a cause of HYP. Tubule maximum (Tm) phosphate per unit glomerular filtration rate decreased and fractional excretion increased with decreasing phosphate quintiles, indicating reduced tubule phosphate reabsorption was responsible for HYP. Urine pH and serum chloride increased with decreasing serum phosphate, suggesting a coordinate change in NHE3 activity. Serum uric acid and Tm uric acid decreased significantly with decreasing serum phosphate, while uric acid excretion did not vary. Conclusion. HYP in SFs results from decreased tubule phosphate reabsorption and, being associated with related changes in other proximal tubule transporters, may arise from alterations in or signaling to PDZ-containing proteins.

Activation and Inhibition of Sodium-Hydrogen Exchanger Is a Mechanism That Links the Pathophysiology and Treatment of Diabetes Mellitus With That of Heart Failure.

The mechanisms underlying the progression of diabetes mellitus and heart failure are closely intertwined, such that worsening of one condition is frequently accompanied by worsening of the other; the degree of clinical acceleration is marked when the 2 coexist. Activation of the sodium-hydrogen exchanger in the heart and vasculature (NHE1 isoform) and the kidneys (NHE3 isoform) may serve as a common mechanism that links both disorders and may underlie their interplay. Insulin insensitivity and adipokine abnormalities (the hallmarks of type 2 diabetes mellitus) are characteristic features of heart failure; conversely, neurohormonal systems activated in heart failure (norepinephrine, angiotensin II, aldosterone, and neprilysin) impair insulin sensitivity and contribute to microvascular disease in diabetes mellitus. Each of these neurohormonal derangements may act through increased activity of both NHE1 and NHE3. Drugs used to treat diabetes mellitus may favorably affect the pathophysiological mechanisms of heart failure by inhibiting either or both NHE isoforms, and drugs used to treat heart failure may have beneficial effects on glucose tolerance and the complications of diabetes mellitus by interfering with the actions of NHE1 and NHE3. The efficacy of NHE inhibitors on the risk of cardiovascular events may be enhanced when heart failure and glucose intolerance coexist and may be attenuated when drugs with NHE inhibitory actions are given concomitantly. Therefore, the sodium-hydrogen exchanger may play a central role in the interplay of diabetes mellitus and heart failure, contribute to the physiological and clinical progression of both diseases, and explain certain drug-drug and drug-disease interactions that have been reported in large-scale randomized clinical trials.

Both NHERF3 and NHERF2 are necessary for multiple aspects of acute regulation of NHE3 by elevated Ca2+, cGMP, and lysophosphatidic acid.

The intestinal epithelial brush border Na+/H+ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its COOH terminus that include the four multi-PDZ domain-containing NHERF family proteins. All bind to NHE3 and take part in different aspects of NHE3 regulation. Because the roles of each NHERF appear to vary on the basis of the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of NHERF3 and NHERF2 in C57BL/6 mouse jejunum using homozygous NHERF2 and NHERF3 knockout mice. NHE3 activity was determined with two-photon microscopy and the dual-emission pH-sensitive dye SNARF-4F. The jejunal apical membrane of NHERF3-null mice appeared similar to wild-type (WT) mice in surface area, microvillus number, and height, which is similar to results previously reported for jejunum of NHERF2-null mice. NHE3 basal activity was not different from WT in either NHERF2- or NHERF3-null jejunum, while d-glucose-stimulated NHE3 activity was reduced in NHERF2, but similar to WT in NHERF3 KO. LPA stimulation and UTP (elevated Ca2+) and cGMP inhibition of NHE3 were markedly reduced in both NHERF2- and NHERF3-null jejunum. Forskolin inhibited NHE3 in NHERF3-null jejunum, but the extent of inhibition was reduced compared with WT. The forskolin inhibition of NHE3 in NHERF2-null mice was too inconsistent to determine whether there was an effect and whether it was altered compared with the WT response. These results demonstrate similar requirement for NHERF2 and NHERF3 in mouse jejunal NHE3 regulation by LPA, Ca2+, and cGMP. The explanation for the similarity is not known but is consistent with involvement of a brush-border NHERF3-NHERF2 heterodimer or sequential NHERF-dependent effects in these aspects of NHE3 regulation. NEW & NOTEWORTHY NHERF2 and NHERF3 are apical membrane multi-PDZ domain-containing proteins that are involved in regulation of intestinal NHE3. This study demonstrates that NHERF2 and NHERF3 have overlapping roles in NHE3 stimulation by LPA and inhibition by elevated Ca2+ and cGMP. These results are consistent with their role being as a NHERF3-NHERF2 heterodimer or via sequential NHERF-dependent signaling steps, and they begin to clarify a role for multiple NHERF proteins in NHE3 regulation.