Sodium-hydrogen Exchanger Isoform Research Articles

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

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

Study of the stability of the biologically active compound N-[2-[4-oxo-3(4H)-quinazolinyl]propionyl]-guanidine using electrophoresis and mass spectrometry

At the Scientific Centre of Innovative Medicines of Volgograd State Medical University, a number of pyrimidine derivatives of guanidine were designed and synthesised to study them as promising inhibitors of sodium-hydrogen exchanger isoform 1 (NHE-1), which can reduce tissue damage during ischemia. N-[2-[4-oxo-3(4H)-quinazolinyl]propionyl]-guanidine (VMA-13-15) proved to be the most pharmacologically valuable compound. When preparing regulatory documentation for a new medicine, one of the quality criteria is the amount of related impurities. They can be technological impurities and degradation products of the active substance. The aim of the research was to study the stability of the biologically active compound N-[2-[4-[4-oxo-3(4H)-quinazolinyl]propionyl]-guanidine. Anticipated degradation products were determined by stress testing, using capillary electrophoresis and mass spectroscopy. The study of the molecule of VMA-13-15 and the use of computer technologies allowed us to predict possible degradation processes. Among them, the most likely to occur is the hydrolysis reaction during thermostating of the solution of the studied compound in an alkaline medium. In silico optimisation of the geometry of the studied structures was carried out by the MM+ molecular mechanics method using HyperChem 8.0.9. The final spatial optimisation of the geometry of the modelled molecules was carried out by the density functional theory method ub3lyp with 6-311G** basis sets. The resulting vibrational analysis of thermodynamic characteristics was carried out in Orca, which allowed us to obtain enthalpy and entropy values for the studied compounds. By capillary electrophoresis, the degradation products of an aqueous solution of the studied substance were analysed by stress tests. They were used to study the effect of thermal treatment on VMA-13-15 in the presence of 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution. We found that the alkaline environment had the greatest effect on the stability of N-[2-[4-oxo-3(4H)-quinazolinyl]propionyl]-guanidine. It was consistent with the results of the computational experiment. We studied the mass spectrum of the degradation product detected during thermostatic treatment in alkaline medium. Based on its molar mass and release time, it was confirmed that the impurity on the chromatograms of VMA-13-15 is unsubstituted quinazolin-4(3H)-one, the initial product of the synthesis

Lack of Renal NHE1 Exacerbates Lithium-Induced Nephrogenic Diabetes Insipidus

The sodium-hydrogen exchanger isoform 1 (NHE1) is a transporter with roles in transepithelial Na+/H+ transport, intracellular pH (pHi), and cell volume regulation. Within the kidney, we localized NHE1 to the basolateral membrane of the distal parts of the nephron and collecting duct (co-labelling with AQP2 for principal cells and H+-ATPase B1 subunit for all 3 types of intercalated cells: type A, B, and non-A-non-B). We have previously shown that lack of NHE3 does not affect Li+ clearance, and other studies have shown that NHE1 transports Li+ preferably compared to NHE3. Therefore we hypothesized that NHE1 plays a critical role in Li+-induced nephrogenic diabetes insipidus. To address this question, we used newly generated mice which lack NHE1 protein in the tubule/collecting duct system (NHE1KS-KO) of the kidney. After baseline measurements, control (n=12) and NHE1KS-KO (n=9) mice were switched to a Li+-containing diet (0.2% LiCl [40 mmol/kg], Harlan Teklad, TD09326) for 4 weeks. Urine osmolality was not different between groups under baseline conditions (control: 2641±178, NHE1KS-KO: 2716±244 mmol/kg). In response to Li+, urine osmolality reached a minimum after 7 days that was sustained until the end of the experimental period. Of note, urine osmolality was ~500 mmol/kg lower in NHE1KS-KO versus control mice (1125±170 versus 475±94 mmol/kg, P<0.05) despite genotypes having comparable plasma Li+ concentrations (0.48±0.1 versus 0.45±0.1 mmol/L). Plasma Na+ concentrations were not significantly different under baseline conditions (149±0.5 versus 148±0.4 mmol/L) but increased to a significantly greater extent in NHE1KS-KO versus control mice (156±1.9 versus 152±0.5 mmol/L, P<0.05). Along those lines, plasma Cl− was significantly greater in NHE1KS-KO versus control mice at the end of the experimental period (117±1.9 versus 111±0.4 mmol/L, P<0.05). Taken together, our data indicate that NHE1 in principal cells plays an important role in the development of nephrogenic diabetes insipidus as well as the development of hypernatremia and hyperchloremia. This work was supported by a VA Merit Review Award IBX004968A (to Dr. Rieg) and a Pilot Project from the USF Microbiomes Institute (to T.R. and J.D.R). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

N123I mutation in the ALV-J receptor-binding domain region enhances viral replication ability by increasing the binding affinity with chNHE1.

The subgroup J avian leukosis virus (ALV-J), a retrovirus, uses its gp85 protein to bind to the receptor, the chicken sodium hydrogen exchanger isoform 1 (chNHE1), facilitating viral invasion. ALV-J is the main epidemic subgroup and shows noteworthy mutations within the receptor-binding domain (RBD) region of gp85, especially in ALV-J layer strains in China. However, the implications of these mutations on viral replication and transmission remain elusive. In this study, the ALV-J layer strain JL08CH3-1 exhibited a more robust replication ability than the prototype strain HPRS103, which is related to variations in the gp85 protein. Notably, the gp85 of JL08CH3-1 demonstrated a heightened binding capacity to chNHE1 compared to HPRS103-gp85 binding. Furthermore, we showed that the specific N123I mutation within gp85 contributed to the enhanced binding capacity of the gp85 protein to chNHE1. Structural analysis indicated that the N123I mutation primarily enhanced the stability of gp85, expanded the interaction interface, and increased the number of hydrogen bonds at the interaction interface to increase the binding capacity between gp85 and chNHE1. We found that the N123I mutation not only improved the viral replication ability of ALV-J but also promoted viral shedding in vivo. These comprehensive data underscore the notion that the N123I mutation increases receptor binding and intensifies viral replication.

Intestinal Permeability, Irritable Bowel Syndrome with Constipation, and the Role of Sodium-Hydrogen Exchanger Isoform 3 (NHE3).

Increased intestinal permeability has been identified as one of the many pathophysiological factors associated with the development of irritable bowel syndrome (IBS), a common disorder of gut-brain interaction. The layer of epithelial cells that lines the intestine is permeable to a limited degree, and the amount of paracellular permeability is tightly controlled to enable the absorption of ions, nutrients, and water from the lumen. Increased intestinal permeability to macromolecules can be triggered by a variety of insults, including infections, toxins from food poisoning, or allergens, which in turn cause an inflammatory response and are associated with abdominal pain in patients with IBS. This review article discusses increased intestinal permeability in IBS, focusing on IBS with constipation (IBS-C) through the lens of a patient case with a reported prior diagnosis of "leaky gut syndrome" upon initial contact with a gastrointestinal specialist. We review advantages and disadvantages of several methods of measuring intestinal permeability in patients and discuss when measuring intestinal permeability is appropriate in the therapeutic journey of patients with IBS-C. Furthermore, we discuss a possible mechanism of restoring the intestinal barrier to its healthy state through altering intracellular pH by inhibiting sodium-hydrogen exchanger isoform 3 (NHE3). Tenapanor is a minimally absorbed, small-molecule inhibitor of NHE3 that has been approved by the US Food and Drug Administration for the treatment of IBS-C in adults. Preclinical studies showed that tenapanor may restore the intestinal barrier in IBS-C by affecting the conformation of tight junction proteins via NHE3 inhibition to block the paracellular transport of macromolecules from the intestinal lumen. Testing for increased permeability in patients with IBS-C who experience abdominal pain may help inform the choice of therapeutics and alter patients' misconceptions about "leaky gut syndrome".

Similarities and Distinctions Between Acetazolamide and SGLT2 Inhibitors in Patients With Acute Heart Failure: Key Insights Into ADVOR and EMPULSE.

Both acetazolamide and sodium-glucose cotransporter 2 (SGLT2) inhibitors block sodium reabsorption in the proximal renal tubule primarily through inhibition of sodium hydrogen exchanger isoform 3 (NHE3), but neither SGLT2 inhibitors nor acetazolamide produce a sustained natriuresis due to compensatory upregulation of sodium reabsorption at distal nephron sites. Nevertheless, acetazolamide and SGLT2 inhibitors have been used as adjunctive therapy to loop diuretics in states where NHE3 is upregulated, e.g., acute heart failure. Two randomized controlled trials have been carried out with acetazolamide in acute heart failure (DIURESIS-CHF and ADVOR). In ADVOR, acetazolamide improved physical signs of fluid retention, but this finding could not be explained by the modest observed diuretic effect. Acetazolamide did not produce a natriuresis in the DIURESIS-CHF trial, and in ADVOR, immediate effects on symptoms and body weight were not reported, and the drug had no effect on morbidity or mortality after 90 days. Three randomized controlled trials have been carried out with empagliflozin (EMPAG-HF, EMPA-RESPONSE-AHF and EMPULSE in acute heart failure. The EMPULSE trial did not report effects on diuresis or in changes in physical signs of congestion during the first week of treatment, but in EMPAG-HF and EMPA-RESPONSE-AHF, empagliflozin had no effect of dyspnea, urinary sodium excretion or body weight during the first 4 days. In the EMPULSE trial, empagliflozin improved health status at 15 days and reduced the risk of worsening heart failure events at 90 days, but these effects are similar in magnitude and time course to the early statistical significance on the risk of heart failure hospitalizations achieved within 14-30 days in the major trials of SGLT2 inhibitors in patients with chronic heart failure. Neurohormonal inhibitors produce this early effect in the absence of a diuresis. Additionally, in numerous randomized controlled trials, in-hospital diuretic intensification has not reduced the risk of major heart failure events, even when treatment is sustained. These findings, taken collectively, suggest that any immediate diuretic effects of acetazolamide and SGLT2 inhibitors in acute heart failure are not likely to influence the short- or long-term clinical course of patients This article is protected by copyright. All rights reserved.

Renal NHE1 is required for regulation of intracellular pH in principal cells and type B intercalated cells

The sodium-hydrogen exchanger isoform 1 (NHE1) is a plasma membrane transporter with roles in transepithelial Na+/H+ transport, intracellular pH (pHi) and cell volume regulation. Within the kidney, NHE1 is previously documented to be expressed in all nephron segments. However, recent transcriptomic and proteomic analyses, and our staining results in mice, consistently find no expression of NHE1 in the proximal tubule or thick ascending limb, but in the distal nephron, including distal convoluted tubule, connecting tubule, and collecting duct (principal and intercalated cells, PC and IC, respectively). We hypothesized that basolateral NHE1 in IC (co-labelling with H+-ATPase B1 subunit, labeling all 3 types of IC: type A, B, and non-A-non-B) plays an important role in acid-base homeostasis. Due to lack of a suitable animal model, we generated kidney-specific NHE1 knockout (NHE1KS-KO) mice. NHE1KS-KO mice completely lack renal NHE1 protein expression compared to their control littermates (n=6 male mice/genotype). No differences were observed in body weight, food and water intake (determined in their home cages) or concentrations of blood Na+, K+, Ca2+, pH or HCO3-. Urine analysis showed no significant differences in electrolytes, minerals of pH. To mechanistically determine the role of NHE1 for pHi regulation in IC and PC we used confocal microscopy of acutely-split open cortical collecting ducts. This method allows for the analysis of a vast number of PC/IC simultaneously. To study the role of NHE1 we employed a NH4Cl pre-pulse (40 mM) and measured pHi recovery after switching back to a physiological solution. Intracellular pH recovery in PC (n=171) was significantly slower (~50%) in NHE1KS-KO mice compared to controls (n=85). Similar to PC, pHi recovery in type B IC of NHE1KS-KO mice (n=38) was significantly slower (~35%) compared to controls (n=40). No differences in type A IC were observed between NHE1KS-KO mice (n=35) and controls (n=29). Inhibition of ClC-K2 via NPPB (Cl-channel blocker) was used to confirm cell types: PC did not respond with a change in pHi but NPPB increased pHi in type A and decreased pHi in type B ICs. In summary, our results identify for the first time an important role of NHE1 for pHi regulation particularly in PCs and type B ICs. Funding to Dr. Rieg was provided by the NIDDK grant 1R01DK110621, VA Merit Review Award IBX004968A, AHA Transformational Research Award 19TPA34850116, and NIDDK Diabetic Complications Consortium grants DK076169 and DK115255. DK095029 and DK117865 (both to O. Pochynyuk). Dr. Thomas was supported by an AHA postdoctoral grant (828731), V. Tomilin by an AHA Career Development Award 19CDA34660148. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Kidney and blood pressure regulation-latest evidence for molecular mechanisms.

Hypertension is one of the major health problems leading to the development of cardiovascular diseases. Despite a rapid expansion in global hypertension prevalence, molecular mechanisms leading to hypertension are not fully understood largely due to the complexity of pathogenesis involving several factors. Salt intake is recognized as a leading determinant of blood pressure, since reduced dietary salt intake is related to lower morbidity and mortality, and hypertension in relation to cardiovascular events. Compared with salt-resistant populations, salt-sensitive individuals exhibit high sensitivity in blood pressure responses according to changes in salt intake. In this setting, the kidney plays a major role in the maintenance of blood pressure under the hormonal control of the renin-angiotensin-aldosterone system. In the present review, we summarize the current overview on the molecular mechanisms for modulation of blood pressure associated with renal ion channels/transporters including sodium-hydrogen exchanger isoform 3 (NHE3), Na+-K+-2Cl- cotransporter (NKCC2), sodium-chloride cotransporter (NCC), epithelial sodium channel (ENaC) and pendrin expressed in different nephron segments. In particular, recent studies on experimental animal models with deletion of renal ion channels led to the identification of several crucial physiological mechanisms and molecules involved in hypertension. These findings could further provide a potential for novel therapeutic approaches applicable on human patients with hypertension.

NHE1 as a Target to Block Lung Fibrosis Progression

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial pneumonia of unknown origin. Fibrosis, a wound healing process, occurs when fibroblasts increase proliferation, convert to myofibroblasts, and secrete extracellular matrix (ECM) proteins. As a result, tissue becomes scarred, reducing lung compliance and ultimately leads to organ dysfunction and death. IFP is characterized by profibrotic agonists and alpha‐smooth muscle actin (α‐SMA) expression. TGF‐β, IL‐1β, LPA, 5‐HT are agonists involved in cell migration regulation, proliferation, and cytokine production. Chronic agonists exposure causes α‐SMA expression which stiffens fibrotic tissue alongside ECM proteins. Prior studies have shown that the sodium hydrogen exchanger isoform 1 (NHE1) is a key regulator in cell migration and pathophysiological development of cancer. The characteristics of IFP are similar to that of cancer (i.e., response to growth signals, myofibroblast origin and behavior, altered cellular communications, and intercellular signaling pathways). Due to these similarities, we hypothesize that NHE1 plays a key role in IFP progression. Results show fibroblast to myofibroblast differentiation is induced in the presence of NHE1 and can also be blocked by the NHE1 inhibitor (EIPA) in mammalian hamster cells. Cells treated with agonists showed some to high α‐SMA expression. Cells treated with agonist and EIPA had no expression of α‐SMA (TGF‐β/EIPA: 35.67 ± 1.76; IL‐1β/EIPA: 11.67 ± 2.03, LPA/EIPA: 33.33 ± 2.19, 5HT/EIPA: 30.00 ± 1.15), and is comparable to the untreated control (20.83 ± 3.12). This improves our understanding of NHE1's role in IPF, and how NHE1 can be a target to impede or hopefully reverse myofibroblast differentiation in IPF patients.

Potential Involvement of NHE1 in COVID‐19 Related Pulmonary Damage

In some moderate and severe COVID‐19 infections lung scarring similar to fibrosis have been observed in survivors, many of which have persistent functional and radiographic abnormalities at the initial follow‐up suggesting a possible shared pathobiology between fibrotic lung disease and COVID‐19‐ lung disease. Fibrosis is an inflammatory response to pulmonary infections that activates fibroblasts and produces excessive extracellular matrix proteins that stiffen cells, leading to reduction in lung capacity and breathing deficiency. Several factors involved in the “cytokine storm” also are pre‐fibrotic leading to the advanced disease. This includes interleukin 6 and others. The focus of this study is on sodium hydrogen exchanger isoform 1 (NHE1) that plays a key role in intracellular pH regulation and cell movement and its effect on fibrosis development. Both cytokines IL‐1 and IL‐6 are used to show one measure of cell restructuring and stiffness by measuring stress fiber formation. To examine the role of NHE1 in fibroblast response, both IL‐1 and IL‐6 cells were treated with proinflammatory cytokines in the presence and absence of NHE1 inhibitor (EIPA). This study will provide an initial insight to the relationship between NHE1 and proinflammatory response for future experiments.

NHE1 is Involved in the Cytoskeletal Remodeling and ECM Deposition of Lung Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a progressive lung disease characterized by the deregulation of the wound healing process leaving an accumulation of fibroblasts and scarring tissue. Fibroblasts, under normal conditions, support growth and health of connective tissues and maintain the extracellular matrix (ECM). However, as the disease progresses, increases in profibrotic agonists lead to activation of fibroblasts causing stiffening of the cells, increasing in secretion of ECM proteins in the bronchial. The chronic nature of the disease leads fibroblast differentiating into myofibroblast cells making the tissue less compliant. The affected patient’s alveolar architecture becomes damaged which then leads to lung inflexibility, disruption of gas exchange, respiratory failure, and death. Sodium Hydrogen Exchanger Isoform 1 (NHE1) is an ion transporter protein found in the membrane that mediates cellular pH, cell migration, and cytoskeletal anchoring. NHE1 has also been found to impact the pathophysiological development of cancer and other diseases. Interestingly, a number of signaling pathways which contribute to the activation of tumor cells are also involved in profibrotic behavior in fibroblasts and myofibroblast, the purpose of this study was to investigate if NHE1 worsened IPF and if inhibiting NHE1 would decrease the profibrotic activity of fibrosis. We measured two of the indicators of progressing fibrosis: the remodeling of skeletal proteins through formation of actin stress fibers, and the production and secretion of ECM proteins such as collagen and fibronectin. Actin stress fibers have an important role in cell migration and contractility. To stimulate this formation, cells were treated with three agonists that have all been found to increase fibrotic activity: Transforming Growth Factor‐β (TGF‐β), Serotonin (5HT), and Lysophosphatidic Acid (LPA) in the presence and absence of Ethyl‐Isopropyl Amiloride (EIPA), a NHE1 inhibitor. LPA induced stress fibers 43.94 +/‐ 4.4 % over the control, and both TGF‐β and 5HT also stimulated the formation of stress fibers by 32.08 +/‐ 2.1 and 39.1 +/‐ 4.2 %, respectively, over the control non‐treated cells. In each case, the addition of EIPA blocked stress fibers indicating that all three profibrotic factors required NHE1 activity for cytoskeletal remodeling. Additionally, when any combination of agonists were added in combination or a mixture of all three, it was observed that these combinations seemed to block stress fiber formation in comparison to the control. An initial interpretation hints at a competitive or feed‐back loop to controlling fibrosis. The role of NHE1 in TGF‐ β induced fibroblast‐myofibroblast differentiation is also determined and is identified with expression of alpha smooth muscle actin (α‐SMA) with and without EIPA. Finally, the role of NHE1 in production and secretion of ECM proteins, such as collagen and fibronectin, were investigated to detect changes in these proteins in the presence and absence of agonists and EIPA. This work highlights that NHE1 has a role in supporting profibrotic behavior and may be a novel target to fight IPF.

Sodium hydrogen exchanger (NHE1) palmitoylation and potential functional regulation

AimsDetermine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family. Main methodsNHE1 expressed in native rat tissues or in heterologous cells was assessed for palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with [3H]palmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP. Key findingsIn this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pHi), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pHi which is dependent on the presence of NHE1. SignificancePalmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions.

Functional characterization of the sodium/hydrogen exchanger 8 and its role in proliferation of colonic epithelial cells.

Intestinal NaCl, HCO3-, and fluid absorption are strongly dependent on apical Na+/H+ exchange. The intestine expresses three presumably apical sodium-hydrogen exchanger (NHE) isoforms: NHE2, NHE3, and NHE8. We addressed the role of NHE8 [solute carrier 9A8 (SLC9A8)] and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated versus undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state intracellular pH (pHi), an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pHi as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na+/H+ exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pHi control.

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.

Tubular effects of sodium–glucose cotransporter 2 inhibitors: intended and unintended consequences

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are antihyperglycemic drugs that act by inhibiting renal sodium-glucose cotransport. Here we present new insights into 'off target', or indirect, effects of SGLT2 inhibitors. SGLT2 inhibition causes an acute increase in urinary glucose excretion. In addition to lowering blood glucose, there are several other effects that contribute to the overall beneficial renal and cardiovascular effects. Reabsorption of about 66% of sodium is accomplished in the proximal tubule and dependent on the sodium-hydrogen exchanger isoform 3 (NHE3). SGLT2 colocalizes with NHE3, and high glucose levels reduce NHE3 activity. The proximal tubule is also responsible for the majority of phosphate (Pi) reabsorption. SGLT2 inhibition is associated with increases in plasma Pi, fibroblast growth factor 23 and parathyroid hormone levels in nondiabetics and type 2 diabetes mellitus. Studies in humans identified a urate-lowering effect by SGLT2 inhibition which is possibly mediated by urate transporter 1 (URAT1) and/or glucose transporter member 9 in the proximal tubule. Of note, magnesium levels were also found to increase under SGLT2 inhibition, an effect that was preserved in nondiabetic patients with hypomagnesemia. Cardiorenal effects of SGLT2 inhibition might involve, in addition to direct effects on glucose homeostasis, effects on NHE3, phosphate, urate, and magnesium homeostasis.

Calcineurin homologous protein isoform 2 supports tumor survival via the sodium hydrogen exchanger isoform 1 in non-small cell lung cancer.

Maintaining intracellular pH is crucial for preserving healthy cellular behavior and, when dysregulated, results in increased proliferation, migration, and invasion. The Na+/H+ exchanger isoform 1 is a highly regulated transmembrane antiporter that maintains pH homeostasis by exporting protons in response to intra- and extracellular signals. Activation of Na+/H+ exchanger isoform 1 is exquisitely regulated by the extracellular environment and protein cofactors, including calcineurin B homologous proteins 1 and 2. While Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 1 are ubiquitously expressed, calcineurin B homologous protein 2 shows tissue-specific expression and upregulation in a variety of cancer cells. In addition, calcineurin B homologous protein 2 expression is modulated by tumorigenic extracellular conditions like low nutrients. To understand the role of calcineurin B homologous protein 2 in tumorigenesis and survival in lung cancer, we surveyed existing databases and formed a comprehensive report of Na+/H+ exchanger isoform 1, calcineurin B homologous protein 1, and calcineurin B homologous protein 2 expression in diseased and non-diseased tissues. We show that calcineurin B homologous protein 2 is upregulated during oncogenesis in many adeno and squamous carcinomas. To understand the functional role of calcineurin B homologous protein 2 upregulation, we evaluated the effect of Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 2 depletion on cellular function during cancer progression in situ. Here, we show that calcineurin B homologous protein 2 functions through Na+/H+ exchanger isoform 1 to effect cell proliferation, cell migration, steady-state pHi, and anchorage-independent tumor growth. Finally, we present evidence that calcineurin B homologous protein 2 depletion in vivo has potential to reduce tumor burden in a xenograft model. Together, these data support the tumor-promoting potential of aberrant calcineurin B homologous protein 2 expression and position calcineurin B homologous protein 2 as a potential therapeutic target for the treatment of non-small cell lung cancer.

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

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

NHE1 activity is required for profibrotic agonist activation of cytoskeleton remodeling in lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is an untreatable chronic lung disorder that is characterized by an accumulation of fibroblasts and scarring tissue due to aberrant tissue remodeling. The lung tissue loses flexibility as the disease upsurges and the affected patient’s alveolar architecture is destroyed. This leads to a disruption of gas exchange, eventual respiratory failure, and death. Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound protein that moderates cellular pH, supports cell migration, and regulates cell behavior. NHE1 has also been found to impact the pathophysiology of cancer. Considering that a myriad of signaling pathways that activate tumor cells are also involved in profibrotic activity of fibroblasts and myofibroblasts, the purpose of this study was to investigate if NHE1 exacerbates IPF and if inhibiting NHE1 would decrease the advancing fibrosis. We measured one of the indicators of progressing fibrosis, the remodeling of skeletal proteins, through the formation of actin stress fibers. Actin stress fibers have an important role in cell migration and contractility in fibroblasts. To stimulate this formation, cells were treated with three agonists that increase fibrotic activity: transforming growth factor ‐ β(TGF‐β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of ethyl‐isopropyl amiloride (EIPA). LPA induced stress fibers 43.94+/−4.4 percent over the control, and both TGF‐βand 5HT also stimulated the formation of stress fibers by 32.08+/−2.1 and 39.1+/−4.2 percent over the control, non‐treated cells. In each case, addition of EIPA blocked agonist‐induced stress fiber formation, indicating that all three profibrotic factors require NHE1 activity for cytoskeletal remodeling. Interestingly, we also observed that stimulation in any combination of agonists had seemed to block stress fiber formation in comparison to the control. An initial interpretation indicates that the agonists negatively regulate the others in stimulating stress fiber formation. The role of each agonist in fibroblasts isolated from fibrotic patients and control, non‐diseased tissue are presented. Myofibroblasts are a specialized cell type that contribute to fibrosis. The role of NHE1 in TGF‐βinduced fibroblast‐myofibroblast differentiation is also determined. The marker for myofibroblast differentiation, α‐smooth muscle actin (α‐SMA) was measured with and without EIPA to determine the role of NHE1 in advancing disease. Finally, the role of NHE1 in myofibroblasts was investigated with EIPA and stress fiber changes with agonists. This work highlights for the first time that NHE1 has a role supporting profibrotic factors and may be a novel target or adjuvant to fight IPF.

Phosphorylation and Palmitoylation Dynamically Regulate Sodium Hydrogen Exchanger Isoform 1 (NHE1) Activity: Implications in Health and Disease

The SLC9 family is made up of nine isoforms of sodium hydrogen exchangers (NHE) which regulate pH by exchanging an intracellular proton for an extracellular sodium ion. Isoform 1 (NHE1) is ubiquitously expressed and plays a vital role in multiple cellular processes in addition to regulating pH such as cellular migration and proliferation, control of cell volume, and stress fiber formation. Additionally, NHE1 is a scaffolding protein that organizes protein complexes to regulate various signaling pathways within the cell. Therefore, it is important to understand the factors contributing to NHE1 regulation as maintenance of these critical cellular functions is vital ensure a healthy cell. Regulation of NHE1 occurs onthe large intracellular C‐terminus where binding partners and posttranslational modifications (PTMs) influence the exchanger in both short‐ and long‐term manners. Phosphorylation and palmitoylation are both dynamic reversible posttranslational modifications (PTMs) that regulate proteins to influence critical cellular processes. Multiple studies have shown phosphorylation and palmitoylation can work together in a barcode to regulate proteins and alter cellular functions. Previous work in our lab has shown NHE1 is regulated by palmitoylation and that inhibition of palmitoylation in cells expressing NHE1 decreases NHE1 activity, as well as NHE1 associated cellular functions such as stress fiber formation and cell migration. NHE1 is regulated by phosphorylation of multiple sites through various kinase pathways. To better understand the relationship between palmitoylation and phosphorylation on NHE1 we used various stimuli known to regulate multiple kinase pathways that impact NHE1 phosphorylation and measured the impact on NHE1 palmitoylation. Using lysophosphatidic acid (LPA) and phorbol 12‐myristate (PMA) to increase NHE1 phosphorylation leads to increased palmitoylation of NHE1, while activation of the NHE1 phosphorylation with insulin leads to decreased NHE1 palmitoylation. Furthermore, we have shown that using these stimuli NHE1 activity increases with NHE1 palmitoylation and this can be attenuated by irreversibly inhibiting palmitoylation using 2‐bromopalmitate (2BP). Using this information, we have begun to understand how the presence or absence of palmitoylation and phosphorylation in a coordinated manner contributes to a barcode that dictates a regulatory outcome and cellular response involving the sodium hydrogen exchanger isoform 1 (NHE1). Future studies will target specific kinases involved in these pathways to further explore the mechanism through which palmitoylation is altered.Support or Funding InformationThis work was supported by the University of North Dakota School of Medicine and Health Sciences and P20 GM103442 (to U.N.D.) from the INBRE program of the National Institute of General Medical Sciences. ND EPSCoRDoctoral Dissertation Assistantship UND0023386

Investigation of Post‐Translational Modification on NHE1 Transport Function

The sodium hydrogen exchanger isoform 1 (NHE‐1) is a sodium hydrogen antiporter that regulates intracellular pH (pHi), cell volume, dynamic actin remodeling processes, and coordination of cell migration in mammalian tissues. NHE‐1 is post‐translationally modified by phosphorylation and lipid modification (palmitoylation). The intracellular C‐terminal domain of NHE‐1 is reversibly phosphorylated at multiple sites byseveral kinases including: extracellular signal‐regulated kinase (ERK), protein kinase B (AKT), B‐Raf, p90 Ribosomal S6 Kinase (p90rsk), RhoA Kinase (ROCK), and other protein kinases. We recently identified that NHE‐1 is reversibly palmitoylated (S‐acetylation), in which a 16‐carbon palmitic acid is covalently attached to a cysteine residue on the C‐terminal domain via a thioester linkage. Such modifications are reported to regulate protein trafficking, membrane micro localization, and protein‐protein interactions. Steady‐state pHi assays incubated with pH‐sensitive dye were conducted to measure the change in pHi of Chinese Hamster Lung Fibroblast Cells due to NHE‐1. Investigating agonists and stimulating factors that increase NHE‐1 palmitoylation allows us to determine the impact of palmitoylation on NHE‐1 transport. Palmitoyl‐transferases (PATs) catalyze the lipidation of proteins and are inhibited by 2‐bromo‐palmitate (2BP). The presence of 2BP causes a loss of palmitoylation to occur, effectively inhibiting LPA‐induced NHE‐1 activity by 0.1 pH units. Fetal Bovine Serum (FBS), Lysophosphatidic acid (LPA), Phorbol 12‐myristate 13‐acetate (PMA), and Insulin effectively stimulate NHE‐1 by increasing pHi by 0.1 pH units. In the presence of kinase inhibitors, NHE‐1 activity significantly decreases compared to agonist or serum treatment alone. The impact of the combination of kinase inhibitors and palmitoylation inhibitors on NHE‐1 mediated pHi change was determined. The data supports the hypothesis that palmitoylation and phosphorylation coordinate to subtly modify NHE1 activity in a novel rheostat of the cell.