NHE Inhibitor Research Articles

Decoding empagliflozin\u2019s molecular mechanism of action in heart failure with preserved ejection fraction using artificial intelligence

The use of sodium-glucose co-transporter 2 inhibitors to treat heart failure with preserved ejection fraction (HFpEF) is under investigation in ongoing clinical trials, but the exact mechanism of action is unclear. Here we aimed to use artificial intelligence (AI) to characterize the mechanism of action of empagliflozin in HFpEF at the molecular level. We retrieved information regarding HFpEF pathophysiological motifs and differentially expressed genes/proteins, together with empagliflozin target information and bioflags, from specialized publicly available databases. Artificial neural networks and deep learning AI were used to model the molecular effects of empagliflozin in HFpEF. The model predicted that empagliflozin could reverse 59% of the protein alterations found in HFpEF. The effects of empagliflozin in HFpEF appeared to be predominantly mediated by inhibition of NHE1 (Na+/H+ exchanger 1), with SGLT2 playing a less prominent role. The elucidated molecular mechanism of action had an accuracy of 94%. Empagliflozin’s pharmacological action mainly affected cardiomyocyte oxidative stress modulation, and greatly influenced cardiomyocyte stiffness, myocardial extracellular matrix remodelling, heart concentric hypertrophy, and systemic inflammation. Validation of these in silico data was performed in vivo in patients with HFpEF by measuring the declining plasma concentrations of NOS2, the NLPR3 inflammasome, and TGF-β1 during 12 months of empagliflozin treatment. Using AI modelling, we identified that the main effect of empagliflozin in HFpEF treatment is exerted via NHE1 and is focused on cardiomyocyte oxidative stress modulation. These results support the potential use of empagliflozin in HFpEF.

Sodium Glucose Co-Transporter 2 Inhibitors Ameliorate Endothelium Barrier Dysfunction Induced by Cyclic Stretch through Inhibition of Reactive Oxygen Species.

SGLT-2i’s exert direct anti-inflammatory and anti-oxidative effects on resting endothelial cells. However, endothelial cells are constantly exposed to mechanical forces such as cyclic stretch. Enhanced stretch increases the production of reactive oxygen species (ROS) and thereby impairs endothelial barrier function. We hypothesized that the SGLT-2i’s empagliflozin (EMPA), dapagliflozin (DAPA) and canagliflozin (CANA) exert an anti-oxidative effect and alleviate cyclic stretch-induced endothelial permeability in human coronary artery endothelial cells (HCAECs). HCAECs were pre-incubated with one of the SGLT-2i’s (1 µM EMPA, 1 µM DAPA and 3 µM CANA) for 2 h, followed by 10% stretch for 24 h. HCAECs exposed to 5% stretch were considered as control. Involvement of ROS was measured using N-acetyl-l-cysteine (NAC). The sodium-hydrogen exchanger 1 (NHE1) and NADPH oxidases (NOXs) were inhibited by cariporide, or GKT136901, respectively. Cell permeability and ROS were investigated by fluorescence intensity imaging. Cell permeability and ROS production were increased by 10% stretch; EMPA, DAPA and CANA decreased this effect significantly. Cariporide and GKT136901 inhibited stretch-induced ROS production but neither of them further reduced ROS production when combined with EMPA. SGLT-2i’s improve the barrier dysfunction of HCAECs under enhanced stretch and this effect might be mediated through scavenging of ROS. Anti-oxidative effect of SGLT-2i’s might be partially mediated by inhibition of NHE1 and NOXs.

Oncogenic RAS drives the CRAF-dependent extracellular vesicle uptake mechanism coupled with metastasis.

Oncogenic RAS impacts communication between cancer cells and their microenvironment, but it is unclear how this process influences cellular interactions with extracellular vesicles (EVs). This is important as intercellular EV trafficking plays a key role in cancer invasion and metastasis. Here we report that overexpression of mutant RAS drives the EV internalization switch from endocytosis (in non‐transformed cells) to macropinocytosis (in cancer cells) resulting in enhanced EV uptake. This process depends on the surface proteoglycan, fibronectin and EV engulfment mechanism regulated by CRAF. Both mutant RAS and activated CRAF expression is associated with formation of membrane ruffles to which they colocalize along with actin, sodium‐hydrogen exchangers (NHEs) and phosphorylated myosin phosphatase (pMYPT). RAS‐transformed cells internalize EVs in the vicinity of ruffled structures followed by apparent trafficking to lysosome and degradation. NHE inhibitor (EIPA) suppresses RAS‐driven EV uptake, along with adhesion‐independent clonal growth and experimental metastasis in mice. Thus, EV uptake may represent a targetable step in progression of RAS‐driven cancers.

New Insights into the Critical Importance of Intratubular Na+/H+ Exchanger 3 and Its Potential Therapeutic Implications in Hypertension.

The sodium (Na+) and hydrogen (H+) exchanger 3 (NHE3), known as solute carrier family 9 member 3 (SLC9A3), mediates active transcellular Na+ and bicarbonate reabsorption in the small intestine of the gut and proximal tubules of the kidney. The purpose of this article is to review and discuss recent findings on the critical roles of intestinal and proximal tubule NHE3 in maintaining basal blood pressure (BP) homeostasis and their potential therapeutic implications in the development of angiotensin II (Ang II)-dependent hypertension. Recently, our and other laboratories have generated or used novel genetically modified mouse models with whole-body, kidney-specific, or proximal tubule-specific deletion of NHE3 to determine the critical roles and underlying mechanisms of NHE3 in maintaining basal BP homeostasis and the development of Ang II-induced hypertension at the whole-body, kidney, or proximal tubule levels. The new findings demonstrate that NHE3 contributes to about 10 to 15 mmHg to basal blood pressure levels, and that deletion of NHE3 at the whole-kidney or proximal tubule level, or pharmacological inhibition of NHE3 at the kidney level with an orally absorbable NHE3 inhibitor AVE-0657, attenuates ~ 50% of Ang II-induced hypertension in mice. The results support the proof-of-concept hypothesis that NHE3 plays critical roles in physiologically maintaining normal BP and in the development of Ang II-dependent hypertension. Our results also strongly suggest that NHE3 in the proximal tubules of the kidney may be therapeutically targeted to treat poorly controlled hypertension in humans.

New generation ENaC inhibitors detach cystic fibrosis airway mucus bundles via sodium/hydrogen exchanger inhibition

Cystic fibrosis (CF) is a recessive inherited disease caused by mutations affecting anion transport by the epithelial ion channel cystic fibrosis transmembrane conductance regulator (CFTR). The disease is characterized by mucus accumulation in the airways and intestine, but the major cause of mortality in CF is airway mucus accumulation, leading to bacterial colonization, inflammation and respiratory failure. Several drug targets are under evaluation to alleviate airway mucus obstruction in CF and one of these targets is the epithelial sodium channel ENaC. To explore effects of ENaC inhibitors on mucus properties, we used two model systems to investigate mucus characteristics, mucus attachment in mouse ileum and mucus bundle transport in piglet airways. We quantified mucus attachment in explants from CFTR null (CF) mice and tracheobronchial explants from newborn CFTR null (CF) piglets to evaluate effects of ENaC or sodium/hydrogen exchanger (NHE) inhibitors on mucus attachment. ENaC inhibitors detached mucus in the CF mouse ileum, although the ileum lacks ENaC expression. This effect was mimicked by two NHE inhibitors. Airway mucus bundles were immobile in untreated newborn CF piglets but were detached by the therapeutic drug candidate AZD5634 (patent WO, 2015140527). These results suggest that the ENaC inhibitor AZD5634 causes detachment of CF mucus in the ileum and airway via NHE inhibition and that drug design should focus on NHE instead of ENaC inhibition.

Interaction of NHE1 and TRPA1 Activity in DRG Neurons Isolated from Adult Rats and its Role in Inflammatory Nociception

Transient receptor potential ankyrin 1 (TRPA1) channel is expressed in a subset of nociceptive neurons. This channel integrates several nociceptive signals. Particularly, it is modulated by intracellular pH (pHi). Na+/H+ exchanger 1 (NHE1) contributes to the maintenance of pHi in nociceptors. However, it is currently unknown whether the interaction between TRPA1 and NHE1 contributes to the nociceptive processing. Thus, the purpose of this study was to assess the functional interaction between NHE1 and TRPA1 in small dorsal root ganglion (DRG) neurons from primary culture obtained from adult rats. Moreover, we also evaluated their possible interaction in acute and inflammatory pain. Zoniporide (selective NHE1 inhibitor) reduced pHi and increased intracellular calcium in a concentration-dependent fashion in DRG neurons. Zoniporide and allyl isothiocyanate (AITC, TRPA1 agonist) increased calcium transients in the same DRG neuron, whereas that A-967079 (TRPA1 antagonist) prevented the effect of zoniporide in DRG neurons. Repeated AITC induced TRPA1 desensitization and this effect was prevented by zoniporide. Both NHE1 and TRPA1 were localized at the membrane surface of DRG neurons in culture. Local peripheral zoniporide enhanced AITC-induced pronociception and this effect was prevented by A-967079. Likewise, zoniporide potentiated Complete Freund’s Adjuvant (CFA)-induced hypersensitivity, effect which was prevented by A-967079 in vivo. CFA paw injection increased TRPA1 and decresed NHE1 protein expression in DRG. These results suggest a functional interaction between NHE1 and TRPA1 in DRG neurons in vitro. Moreover, data suggest that this interaction participates in acute and inflamatory pain conditions in vivo.

Investigating the Potential for NHE1 Inhibitors to Function as Adjuvant Therapies in the Treatment of Ovarian Cancer

Each year in the United States over 22,000 women are diagnosed with ovarian cancer leading to more than 14,000 deaths. Differential expression and activity of the Na+-H+ Exchanger Isoform 1 (NHE1) has been demonstrated to regulate cell proliferation in ovarian cancer cells from patients. NHE1 is an 815 amino acid transmembrane transport protein that regulates intracellular pH. NHE1 is hyperactivated in various types of cancer cells leading to a pH inversion that supports both cell proliferation and invasion. A significant issue in ovarian cancer therapy is the development of drug resistance and disease recurrence. In an effort to combat these outcomes, it is hypothesized that combining common ovarian cancer therapeutic agents along with compounds that directly inhibit NHE1 activation will enhance the ability of the therapeutic agents to slow ovarian cancer progression. Two ovarian adenocarcinoma cell lines; CAOV-3 and SKOV-3 are used in this research. Utilizing a proliferation assay, the impact of the ovarian cancer therapeutics, Rucaparib, Olaparib, Carboplatin and Cisplatin, were assessed in the presence and absence of the NHE1 inhibitors Cariporide and Ethylisopropyl Amiloride (EIPA). Dose response curves have been generated for each therapeutic (mean ± SE n=36) to evaluate the impact of combination therapies and supplement IC50 and percent reduction values for each pair. One example includes a 46% reduction in IC50 value for carboplatin alone (47.09 mM) as compared to carboplatin plus Cariporide (25.28 mM) in CAOV-3 cells. Reductions in IC50 values for each of the therapeutics in combination with NHE1 inhibitors ranging from 24% to 92% were seen in both cell lines. This work has been expanded to a three-dimensional environment, investigating the impact of combination therapies in a spheroid assay. To begin to identify a mechanism for NHE1 involvement in ovarian cancer progression and treatment, a steady-state intracellular pH was performed. It has been shown that when chemotherapeutics are paired with NHE1 inhibitors, a synergistic effect is exhibited in which the combination is more proficient at reducing cancer growth than either compound can alone. To confirm this relationship, NHE1 knockout cell lines are being created via CRISPR-Cas9 gene editing. If this relationship is validated, it could allow a reduction in the required drug dosage while maintaining efficacy and hopefully reducing harmful side effects in patients. Our research indicates that the inhibition of NHE1 in combination with standard chemotherapeutics may serve as a potential therapeutic target in ovarian cancer treatment.

Evaluating the Inhibition of the Na + ‐H + exchanger Isoform 1 (NHE1) and Kinases that Activate NHE1 as a Potential Combination Therapies for the Treatment of Ovarian Cancer with Paclitaxel

Every year in the United States there are over 22,000 newly diagnosed cases of ovarian cancer leading to over 14,000 deaths. Ovarian Cancer is the fifth leading cause of cancerous deaths among women. Due to that advanced stage of cancer progression at the time of diagnosis treatment is typically challenging and frequently accompanied by drug resistance and disease recurrence. Increased expression and activation of NHE1 has been shown to regulate cell proliferation in cancer cells from ovarian cancer patients. NHE1 is an 815 amino acid transmembrane protein that regulates intracellular pH. NHE1 hyperactivity is involved in the pH inversion which creates an optimal environment for cancer cell proliferation and invasion. In an effort to combat this disease recurrence and drug resistance, it is hypothesized that combining the chemotherapeutic paclitaxel with inhibitors that alter NHE1 function there will be a synergistic effect that slows ovarian cancer progression. Paclitaxel is a secondary treatment for ovarian cancer. It is a taxane based chemotherapeutic that acts as a microtubule stabilizer that affects the cells in the G2 and M phases of cell growth. In these experiments a direct inhibitor of NHE1, Ethylisopropylamiloride, is used. Additionally, we evaluate BI-D1870, the inhibitor of Ribosomal S6 Kinase (Rsk). Rsk is activated in ovarian cancer and has the ability to stimulate NHE1 activity. The ability to develop a synergistic effect was assessed using a proliferation assay in two ovarian cancer cell lines CAOV-3 and SKOV-3. Proliferation assays were run with paclitaxel alone and then with both paclitaxel and one of the inhibitors. The IC50 values were then determined for paclitaxel alone and in combination with the inhibitor. For example, in SKOV-3 Cells, the IC50 for paclitaxel was 0.086 µM. When combined with BI-D 1870 the IC50 for paclitaxel decreased to 0.12 µM, an 86% reduction. These data indicate that inhibitors of NHE1 or NHE1 activation may be combined with paclitaxel to improve the efficacy of ovarian cancer treatment.

Empagliflozin Downregulates Renal NHE3 Activity and NaPi‐2 Expression and Reduces Blood Pressure in Hypertensive Rats

Sodium-glucose cotransporter 2 (SGLT2) inhibitors, also known as gliflozins, can lower blood pressure (BP) and reduce cardiovascular mortality in hypertensive patients with type 2 diabetes. However, the molecular mechanisms underlying the antihypertensive effects of the SGLT2 inhibitors have not been elucidated. This study tested the hypothesis that empagliflozin, an SGLT2 inhibitor, reduces BP of nondiabetic hypertensive rats and that this effect may be associated with altered expression and/or covalent modification of apical sodium transporters in the renal proximal tubule. To this end, spontaneously hypertensive rats (SHR) and normotensive Wistar rats (12-14 weeks of age) were treated for 2 weeks with empagliflozin 10 mg/kg/day or vehicle (water) orally. BP was measured non-invasively (tail-cuff plethysmography) and invasively (arterial catheterization). Proximal tubule Na+/H+ exchanger isoform 3 (NHE3) activity was assessed by in vivo stationary microperfusion. NHE3 phosphorylated at serine 552 (pNHE3-552), total NHE3 and Na+-dependent phosphate cotransporter type II (NaPi-2) expression in the renal cortex were evaluated by immunoblotting. We found that vehicle-treated SHR exhibited a small increase in BP (9±2 mmHg), whereas empagliflozin reduced BP of hypertensive rats (-10±2 mmHg). No changes in BP were observed in Wistar rats. The in vivo stationary microperfusion analyses showed that empagliflozin inhibited the activity of NHE3 in both hypertensive and normotensive rats. Inhibition of NHE3 activity by empagliflozin was not accompanied by differences in the levels of pNHE3-552 or total NHE3 expression in the renal cortex. On the other hand, NaPi-2 expression was upregulated in the renal cortex of vehicle-treated SHR (226±18 vs. 100±5% in vehicle-treated Wistar rats, p< 0.0001), and treatment with empagliflozin significantly reduced it (163±11 vs. 226±18%, p< 0.01). Renal cortical NaPi-2 abundance was not affected by empagliflozin in normotensive rats. These findings demonstrate empagliflozin lowers blood pressure in nondiabetic hypertensive rats. Moreover, it shows that SGLT2 inhibition may reduce proximal tubule sodium reabsorption via inhibition of NHE3 and downregulation of NaPi-2. Future studies are necessary to evaluate the impact of SGLT2 inhibitors in the distal nephron and its association with the antihypertensive effects of the gliflozins.

NHE1 exacerbates profibrotic activity in idiopathic pulmonary fibrosis and post‐COVID‐19 infection

Idiopathic pulmonary fibrosis (IPF) is a destructive, aging-related pulmonary disease characterized by formation of lung scar tissue. Like other coronaviruses, SARS-COV-2 leads to long term lung damage in some patients in the form of lung fibrosis. In either disease, aggregation of fibroblasts, cell differentiation and excessive tissue remodeling causes the lung tissue to lose lung compliance and oxygen exchange.Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound transport protein that functions to support cell movement, regulate cell activity, and balance intracellular pH. A multitude of signaling pathways involved in the regulating of NHE1 function are also involved in the profibrotic activity of fibroblasts and myofibroblasts; therefore we investigated if NHE1 is involved in profibrotic activity and tissue damage associated with IPF and COVID-19 infection. Because NHE1 is key in the remodeling of actin stress fibers and is involved in stiffness of both disorders, we first investigated the impact of NHE1 on stress fiber formation using agonists involved with both fibrosis and COVID response. Lung fibroblasts were treated with three agonists: transforming growth factor-β (TGF-β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of an NHE1 inhibitor, ethyl-isopropyl amiloride (EIPA). LPA induced stress fiber formation 65.40+/-3.3 percent over the control, and both 5HT and TGF-β also stimulated the formation of stress fibers by 50.27+/−5.7 and 50.91+/−1.4 percent over the untreated cells. In each case, inhibiting NHE1 with EIPA blocked agonist-activated stress fiber formation, delineating that all three profibrotic factors need NHE1 activity for cytoskeletal remodeling. Another indicator of fibrosis is the fibroblast to myofibroblast transdifferentiation. Myofibroblasts are apoptosis-resistant cells that excessively deposit extracellular matrix proteins; the build up of these cells further compromises the structural integrity of the alveoli and contributes to lung dysfunction. TGF- β, a proinflammatory cytokine, significantly contributes to profibrotic activity by stimulating both fibroblast-myofibroblast transition and excess collagen production. Cells treated with TGF-β to induce cell differentiation showed delayed or significantly reduced myofibroblast transformation in the presence of pharmacological inhibitors of NHE1. Specifically, expression of α-smooth muscle actin (α-SMA), the marker for myofibroblast differentiation, was diminished in each CCL39, LL29, and WI38 cell lines by at least 30 percent and a several day delay of the marker's expression. This study demonstrates the potential for NHE1 as a novel target to fight IPF and lung fibrosis in post-COVID-19 infection.

Investigating the Role of NHE1 in the Regulation of Changes in Cellular Functions Related to the Development of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by the thickening, stiffening, and scaring of tissue within the lungs. Affected individuals develop shortness of breath and the progressive reduction of respiratory function. Fibrosis ultimately results in low blood flow and hypoxia in the tissue, a microenvironment which is closely associated with the upregulation of the sodium-hydrogen exchanger isoform 1 (NHE1). NHE1 is a ubiquitously expressed protein that exchanges one extracellular Na+ for one intracellular H+. This regulates intracellular pH and cell progression through the cell cycle. When NHE1 is upregulated it supports increased cell proliferation and migration, indicating that it may play a vital role in the behavior of fibroblasts in the development of idiopathic pulmonary fibrosis. We hypothesized that inhibition of NHE1 activation would decrease cell progression towards a fibrotic phenotype in lung fibroblasts. For this assessment, we compared differences in cellular behavior in three cell lines to evaluate the role of NHE1 in these processes. LL29 cells are human fibroblasts derived from a patient diagnosed with idiopathic pulmonary fibrosis; WI-38 cells which are derived from healthy lung tissue; and PSN cells, which are derived from Chinese hamster lung fibroblasts and express only human NHE1.Transforming growth factor beta 1 (TGF-b1), Lysophosphatidic Acid (LPA) and Serotonin (5-hydroxytryptamine) were evaluated for their ability to alter cell proliferation, stress fiber formation, and myofibroblast differentiation in all three cell lines. TGF-b1 is a cytokine that is a growth factor implicated in the pathogenesis of pulmonary fibrosis and is involved in differentiation, proliferation, and matrix production of fibroblasts. Serotonin (5-HT) is a signaling molecule that binds to G-protein coupled receptors supporting the increase in TGF-b1 and its effects in cells. This hormone is involved in wound healing and stimulating inflammation. LPA is a lipid signaling molecule that plays a role in mediating inflammation and activating TGF-b1 signaling. All experiments were performed in the presence and absence of Ethylisopropylamiloride (EIPA), a potent inhibitor of NHE1, to evaluate the role of NHE1 in fibrotic transformation. In PSN cells, stimulation with 10 µM LPA increased stress fiber formation over 2-fold and this stimulation was absent in cells treated with 10 µM EIPA indicating a role for NHE1 in the regulation of stress fiber formation and related cellular processes.

Na+/H+ exchangers differentially contribute to midgut fluid sodium and proton concentration in the sea urchin larva.

Regulation of ionic composition and pH is a requisite of all digestive systems in the animal kingdom. Larval stages of the marine superphylum Ambulacraria, including echinoderms and hemichordates, were demonstrated to have highly alkaline conditions in their midgut with the underlying epithelial transport mechanisms being largely unknown. Using ion-selective microelectrodes, the present study demonstrated that pluteus larvae of the purple sea urchin have highly alkaline pH (pH∼9) and low [Na+] (∼120 mmol l-1) in their midgut fluids, compared with the ionic composition of the surrounding seawater. We pharmacologically investigated the role of Na+/H+ exchangers (NHE) in intracellular pH regulation and midgut proton and sodium maintenance using the NHE inhibitor 5-(n-ethyl-n-isopropyl)amiloride (EIPA). Basolateral EIPA application decreased midgut pH while luminal application via micro-injections increased midgut [Na+], without affecting pH. Immunohistochemical analysis demonstrated a luminal localization of NHE-2 (SpSlc9a2) in the midgut epithelium. Specific knockdown of spslc9a2 using Vivo-Morpholinos led to an increase in midgut [Na+] without affecting pH. Acute acidification experiments in combination with quantitative PCR analysis and measurements of midgut pH and [Na+] identified two other NHE isoforms, Spslc9a7 and SpSlc9a8, which potentially contribute to the regulation of [Na+] and pH in midgut fluids. This work provides new insights into ion regulatory mechanisms in the midgut epithelium of sea urchin larvae. The involvement of NHEs in regulating pH and Na+ balance in midgut fluids shows conserved features of insect and vertebrate digestive systems and may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.

Inhibition of Na+/H+ exchanger modulates microglial activation and scar formation following microelectrode implantation

Objective. Intracortical microelectrodes are an important tool for neuroscience research and have great potential for clinical use. However, the use of microelectrode arrays to treat neurological disorders and control prosthetics is limited by biological challenges such as glial scarring, which can impair chronic recording performance. Microglia activation is an early and prominent contributor to glial scarring. After insertion of an intracortical microelectrode, nearby microglia transition into a state of activation, migrate, and encapsulate the device. Na+/H+ exchanger isoform-1 (NHE-1) is involved in various microglial functions, including their polarity and motility, and has been implicated in pro-inflammatory responses to tissue injury. HOE-642 (cariporide) is an inhibitor of NHE-1 and has been shown to depress microglial activation and inflammatory response in brain injury models. Approach. In this study, the effects of HOE-642 treatment on microglial interactions to intracortical microelectrodes was evaluated using two-photon microscopy in vivo. Main results. The rate at which microglia processes and soma migrate in response to electrode implantation was unaffected by HOE-642 administration. However, HOE-642 administration effectively reduced the radius of microglia activation at 72 h post-implantation from 222.2 µm to 177.9 µm. Furthermore, treatment with HOE-642 significantly reduced microglial encapsulation of implanted devices at 5 h post-insertion from 50.7 ± 6.0% to 8.9 ± 6.1%, which suggests an NHE-1-specific mechanism mediating microglia reactivity and gliosis during implantation injury. Significance. This study implicates NHE-1 as a potential target of interest in microglial reactivity and HOE-642 as a potential treatment to attenuate the glial response and scar formation around implanted intracortical microelectrodes.

Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective Inhibitors

The K+-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane pH that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine 24 uPA IC50 = 175 nM, NHE1 IC50 = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine 26 uPA IC50 = 86 nM, NHE1 IC50 = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino (29 NHE1 IC50 = 129 nM, uPA IC50 = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) (30 NHE1 IC50 = 85 nM, uPA IC50 = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.

Abstract P761: Reducing Reactive Astrocytes Improves Cognitive Function in a Mouse Model of Vascular Cognitive Impairment and Dementia

Inflammation and reactive astrocytes are pathologic features in vascular cognitive impairment and dementia (VCID). However, there are no effective therapies to reduce chronic hypoperfusion-induced reactive astrocytes and neuroinflammation and cognitive deficits in VCID. Activation of Na + /H + exchanger 1 (NHE1) in astrocytes mediates Na + influx in exchange of H + efflux, causing hypertrophy and swelling of reactive astrocytes after acute brain injury. Whether pharmacological blockade of NHE1 protein can reduce reactive astrocytes and improve cognitive deficits remains untested. Bilateral carotid artery stenosis (BCAS) with microcoils as a murine VCID model was induced in adult male C57BL/6 mice. BCAS procedures led to sustained reduction (30-40%) in cerebral blood flow (CBF) and deficits in special working memory by 30 days post-surgery (p &lt; 0.05). Compared with sham control mice, BCAS mice displayed demyelination in corpus callosum (CC) and external capsule (EC) at 30 days post-surgery (p &lt; 0.05). There were significantly increased GFAP + astrocytes and Iba1 + microglia in CC, EC and hippocampus in BCAS brain at 30 days post-surgery (p &lt; 0.05). Interestingly, those GFAP + astrocytes and Iba1 + microglia exhibited elevated NHE1 protein expression in these brain regions (p &lt; 0.05). These results suggest that NHE1 protein may play a role in chronic hypoperfusion-induced reactive astrocyte transformation and microglia activation. We administered saline vehicle (Veh) or NHE1 inhibitor HOE642 during 30 days post-BCAS (via either i.p. or osmotic mini-pump). The Veh- and HOE642-treated BCAS mice caused similar reduction in CBF. Compared to the Veh controls, both HOE642 treatment regimens significantly improved cognitive performance at 30 days post-surgery (p &lt; 0.05). Causative relationship between blockade of NHE1 protein activation and reducing glia-mediated inflammation and demyelination are under investigation by immunostaining assessments and ex-vivo MRI DTI analysis. Taken together, our study suggests that elevation of NHE1 protein activity plays a role in glial activation in the murine VCID model. Pharmacological blockade of NHE1 protein shows efficacy in reducing cerebral hypoperfusion-induced chronic brain injury and cognitive impairment.

Myocardial Impact of NHE1 Regulation by Sildenafil.

The cardiac Na+/H+ exchanger (NHE1) is a membrane glycoprotein fundamental for proper cell functioning due its multiple housekeeping tasks, including regulation of intracellular pH, Na+ concentration, and cell volume. In the heart, hyperactivation of NHE1 has been linked to the development of different pathologies. Several studies in animal models that reproduce the deleterious effects of ischemia/reperfusion injury or cardiac hypertrophy have conclusively demonstrated that NHE1 inhibition provides cardioprotection. Unfortunately, NHE1 inhibitors failed to reproduce these effects in the clinical arena. The reasons for those discrepancies are not apparent yet. However, a reasonable clue to consider would be that drugs that completely abolish the exchanger activity, including that its essential housekeeping function may not be the best therapeutic approach. Therefore, interventions tending to specifically reduce its hyperactive state without affecting its basal activity emerge as a novel potential gold standard. In this regard, a promising goal seems to be the modulation of the phosphorylation state of the cytosolic tail of the exchanger. Recent own experiments demonstrated that Sildenafil, a phosphodiesterase 5A inhibitor drug that has been widely used for the treatment of erectile dysfunction is able to decrease NHE1 phosphorylation, and hence reduce its hyperactivity. In connection, growing evidence demonstrates cardioprotective properties of Sildenafil against different cardiac pathologies, with the distinctive characteristic of directly affecting cardiac tissue without altering blood pressure. This mini-review was aimed to focus on the regulation of NHE1 activity by Sildenafil. For this purpose, experimental data reporting Sildenafil effects in different animal models of heart disease will be discussed.

Integrin-Linked Kinase Links Integrin Activation to Invadopodia Function and Invasion via the p(T567)-Ezrin/NHERF1/NHE1 Pathway.

Tumor cell invasion depends largely on degradation of the extracellular matrix (ECM) by protease-rich structures called invadopodia, whose formation and activity requires the convergence of signaling pathways engaged in cell adhesion, actin assembly, membrane regulation and ECM proteolysis. It is known that β1-integrin stimulates invadopodia function through an invadopodial p(T567)-ezrin/NHERF1/NHE1 signal complex that regulates NHE1-driven invadopodia proteolytic activity and invasion. However, the link between β1-integrin and this signaling complex is unknown. In this study, in metastatic breast (MDA-MB-231) and prostate (PC-3) cancer cells, we report that integrin-linked kinase (ILK) integrates β1-integrin with this signaling complex to regulate invadopodia activity and invasion. Proximity ligation assay experiments demonstrate that, in invadopodia, ILK associates with β1-integrin, NHE1 and the scaffold proteins p(T567)-ezrin and NHERF1. Activation of β1-integrin increased both invasion and invadopodia activity, which were specifically blocked by inhibition of either NHE1 or ILK. We conclude that ILK integrates β1-integrin with the ECM proteolytic/invasion signal module to induce NHE1-driven invadopodial ECM proteolysis and cell invasion.

Neuroprotective effect of Na+ /H+ exchangers isoform-1 inactivation against 6-hydroxydopamine-induced mitochondrial dysfunction and neuronal apoptosis in Parkinson's disease models.

Parkinson's disease (PD) is a disabling neurodegenerative disease mainly caused by degeneration of mesencephalic dopaminergic neurons in the substantia nigra pars compacta (SNpc). The neuroprotective role of Na+ /H+ exchangers isoform-1 (NHE1) inactivation in cerebral ischemic damage has been elucidated. The current study aimed to investigate the impacts of NHE1 in PD. In this study, 6-hydroxydopamine (6-OHDA)-induced PD rat models were established to attempt to illuminate the role and underlying mechanisms of NHE1 in SNpc neurons of PD. Meanwhile, nerve growth factor-stimulated PC12 cells followed by 6-OHDA treatment was used to mimic PD in vitro. Results showed that the protein levels of NHE1 were significantly increased in the SNpc neurons of rats and differentiated PC12 cells after 6-OHDA treatment. Inactivation of NHE1 with chemical inhibitor HOE642 suppressed SNpc neuronal loss and NHE1 expression in PD rats. The overlays of tyrosine hydroxylase and NHE1 displayed that NHE1 expression was not colocalized but closely associated with TH. Besides, treatment with HOE642 relieved the dyskinesia, mitochondrial dysfunction, and neuronal apoptosis. Further in vitro evidence confirmed that inhibition of NHE1 by genetic-knockdown prevented mitochondrial dysfunction and apoptosis. Our study represents the first experimental evidence of a potential role for NHE1 in the pathogenesis of PD.

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

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

Cardiomyocyte Na+/H+ Exchanger-1 Activity Is Reduced in Hypoxia.

Fully-activated Na+/H+ exchanger-1 (NHE1) generates the cardiomyocyte's largest trans-membrane extrusion of H+ ions for an equimolar influx of Na+ ions. This has the desirable effect of clearing excess intracellular acidity, but comes at a large energetic premium because the exchanged Na+ ions must ultimately be extruded by the sodium pump, a process that consumes the majority of the heart's non-contractile ATP. We hypothesize that the state of NHE1 activation depends on metabolic resources, which become limiting in periods of myocardial hypoxia. To test this functionally, NHE1 activity was measured in response to in vitro and in vivo hypoxic treatments. NHE1 flux was interrogated as a function of intracellular pH by fluorescence imaging of rodent ventricular myocytes loaded with pH-sensitive dyes BCECF or cSNARF1. Anoxic superfusates promptly inhibited NHE1, tracking the time-course of mitochondrial depolarization. Mass spectrometry of NHE1 immuno-precipitated from Langendorff-perfused anoxic hearts identified Tyr-581 dephosphorylation and Tyr-561 phosphorylation. The latter residue is part of the domain that interacts with phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid that becomes depleted under metabolic inhibition. Tyr-561 phosphorylation is expected to electrostatically weaken this activatory interaction. To test if a period of hypoxia produces a persistent inhibition of NHE1, measurements under normoxia were performed on myocytes that had been incubated in 2% O2 for 4 h. NHE1 activity remained inhibited, but the effect was ablated in the presence of Dasatinib, an inhibitor of Abl/Src-family tyrosine kinases. Chronic tissue hypoxia in vivo, attained in a mouse model of anemic hypoxia, also resulted in persistently slower NHE1. In summary, we show that NHE1 responds to oxygen, a physiologically-relevant metabolic regulator, ostensibly to divert ATP for contraction. We describe a novel mechanism of NHE1 inhibition that may be relevant in cardiac disorders featuring altered oxygen metabolism, such as myocardial ischemia and reperfusion injury.