NHE Activity Research Articles

Metformin Inhibits Na+/H+ Exchanger NHE3 Resulting in Intestinal Water Loss.

Glycemic control is the key to the management of type 2 diabetes. Metformin is an effective, widely used drug for controlling plasma glucose levels in diabetes, but it is often the culprit of gastrointestinal adverse effects such as abdominal pain, nausea, indigestion, vomiting, and diarrhea. Diarrhea is a complex disease and altered intestinal transport of electrolytes and fluid is a common cause of diarrhea. Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and our previous study has demonstrated that decreased NHE3 contributes to diarrhea associated with type 1 diabetes. The goal of this study is to investigate whether metformin regulates NHE3 and inhibition of NHE3 contributes to metformin-induced diarrhea. We first determined whether metformin alters intestinal water loss, the hallmark of diarrhea, in type 2 diabetic db/db mice. We found that metformin decreased intestinal water absorption mediated by NHE3. Metformin increased fecal water content although mice did not develop watery diarrhea. To determine the mechanism of metformin-mediated regulation of NHE3, we used intestinal epithelial cells. Metformin inhibited NHE3 activity and the effect of metformin on NHE3 was mimicked by a 5′-AMP-activated protein kinase (AMPK) activator and blocked by pharmacological inhibition of AMPK. Metformin increased phosphorylation and ubiquitination of NHE3, resulting in retrieval of NHE3 from the plasma membrane. Previous studies have demonstrated the role of neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2) in regulation of human NHE3. Silencing of Nedd4-2 mitigated NHE3 inhibition and ubiquitination by metformin. Our findings suggest that metformin-induced diarrhea in type 2 diabetes is in part caused by reduced Na+ and water absorption that is associated with NHE3 inhibition, probably by AMPK.

NOX activation in reactive astrocytes regulates astrocytic LCN2 expression and neurodegeneration

Reactive astrocytes (RA) secrete lipocalin-2 (LCN2) glycoprotein that regulates diverse cellular processes including cell death/survival, inflammation, iron delivery and cell differentiation. Elevated levels of LCN2 are considered as a biomarker of brain injury, however, the underlying regulatory mechanisms of its expression and release are not well understood. In this study, we investigated the role of astrocytic Na+/H+ exchanger 1 (NHE1) in regulating reactive astrocyte LCN2 secretion and neurodegeneration after stroke. Astrocyte specific deletion of Nhe1 in Gfap-CreER+/−;Nhe1f/f mice reduced astrogliosis and astrocytic LCN2 and GFAP expression, which was associated with reduced loss of NeuN+ and GRP78+ neurons in stroke brains. In vitro ischemia in astrocyte cultures triggered a significant increase of secreted LCN2 in astrocytic exosomes, which caused neuronal cell death and neurodegeneration. Inhibition of NHE1 activity during in vitro ischemia with its potent inhibitor HOE642 significantly reduced astrocytic LCN2+ exosome secretion. In elucidating the cellular mechanisms, we found that stroke triggered activation of NADPH oxidase (NOX)-NF-κB signaling and ROS-mediated LCN2 expression. Inhibition of astrocytic NHE1 activity attenuated NOX signaling and LCN2-mediated neuronal apoptosis and neurite degeneration. Our findings demonstrate for the first time that RA use NOX signaling to stimulate LCN2 expression and secretion. Blocking astrocytic NHE1 activity is beneficial to reduce LCN2-mediated neurotoxicity after stroke.

Empagliflozin reduces oxidative stress through inhibition of the novel inflammation/NHE/[Na+]c/ROS-pathway in human endothelial cells

Inflammation causing oxidative stress in endothelial cells contributes to heart failure development. Sodium/glucose cotransporter 2 inhibitors (SGLT2i’s) were shown to reduce heart failure hospitalization and oxidative stress. However, how inflammation causes oxidative stress in endothelial cells, and how SGLT2i’s can reduce this is unknown. Here we hypothesized that 1) TNF-α activates the Na+/H+ exchanger (NHE) and raises cytoplasmatic Na+ ([Na+]c), 2) increased [Na+]c causes reactive oxygen species (ROS) production, and 3) empagliflozin (EMPA) reduces inflammation-induced ROS through NHE inhibition and lowering of [Na+]c in human endothelial cells. Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) were incubated with vehicle (V), 10 ng/ml TNF-α, 1 µM EMPA or the NHE inhibitor Cariporide (CARI, 10 µM) and NHE activity, intracellular [Na+]c and ROS were analyzed. TNF-α enhanced NHE activity in HCAECs and HUVECs by 92% (p < 0.01) and 51% (p < 0.05), respectively, and increased [Na+]c from 8.2 ± 1.6 to 11.2 ± 0.1 mM (p < 0.05) in HCAECs. Increasing [Na+]c by ouabain elevated ROS generation in both HCAECs and HUVECs. EMPA inhibited NHE activity in HCAECs and in HUVECs. EMPA concomitantly lowered [Na+]c in both cell types. In both cell types, TNF α-induced ROS was lowered by EMPA or CARI, with no further ROS lowering by EMPA in the presence of CARI, indicating EMPA attenuated ROS through NHE inhibition. In conclusion, inflammation induces oxidative stress in human endothelial cells through NHE activation causing elevations in [Na+]c, a process that is inhibited by EMPA through NHE inhibition.

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

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

Potential Therapeutic Effect of Citronellal on Diabetic Cardiomyopathy in Experimental Rats.

Diabetic cardiomyopathy (DCM), a cardiovascular complication of patients with diabetes, is a special cardiomyopathy that is independent of coronary heart disease, hypertension, and valvular disease. Citronellal (CT) is a monoterpene compound generated by the secondary metabolism of plants. In this work, the therapeutic effect and mechanism of CT in DCM were investigated. Experimental diabetic rat models were constructed through a high-fat and high-carbohydrate diet combined with low-dosage streptozotocin (STZ) treatment. CT was intragastrically administered at the dosage of 150 mg/kg/day. The cardiac functions of the rats were evaluated via cardiac Doppler ultrasound. Changes in myocardial structure were analyzed through histopathology. Changes in the representative indices of oxidative stress, namely, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected on the basis of a biochemical test. Related protein levels were assayed via immunofluorescence and Western blot analyses. The DCM rats in the nontreatment group experienced diastolic and systolic dysfunctions, associated with myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Moreover, this condition was concurrent with metabolic disorders, the degradation of SOD activity in myocardial tissues, the increase in MDA content, the abnormal activation of sodium–hydrogen exchanger 1 (NHE1), and the aggravation of cell apoptosis (Bax levels were elevated, whereas Bcl-2 levels decreased). Myocardial hypertrophy, fibrosis, oxidative stress, and cell apoptosis were obviously inhibited after treatment with CT (150 mg/kg/day). The abnormal activation of NHE1 was recovered under the action of CT. Our study results showed that CT might play a protective role in the treatment of DCM by repressing the abnormal activation of NHE1.

Decreased NHE3 activity in intestinal epithelial cells in TGEV and PEDV-induced piglet diarrhea

Transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED) are highly transmissible intestinal infections caused by transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV), respectively. They are clinically associated with vomiting, diarrhea, and dehydration in piglets. An imbalance in Na+ uptake by intestinal epithelial cells causes TGEV/PEDV-induced diarrhea. However, the mechanism by which TGEV/PEDV-infection in piglets causes Na+ imbalance diarrhea has not been elucidated. In the present study, we demonstrated that specific inhibition of NHE3 activity caused small intestinal bulging, intestinal wall thinning and severe diarrhea in piglets, consistent with the signs of TGEV/PEDV infection. This study further elucidated the role of NHE3 in TGEV/PEDV-induced diarrhea. In this study, small intestinal epithelial cells (IPEC-J2) were used as a model of infection. The results showed that TGEV/PEDV infection reduced NHE3 activity and Na+ uptake in IPEC-J2 cells. Further studies revealed that the use of NHE3-specific inhibitors could reduce the amount of cell membrane NHE3, thereby decreasing Na+ uptake and ultimately leading to diarrhea. Transcriptomic studies performed on obtained jejunal tissues were also consistent with pre-laboratory results. This study will provide a basis for understanding Na+ imbalance diarrhea caused by TGEV/PEDV, as well as for elucidating the diarrheal pathogenesis of other members of α-animal coronaviruses.

Empagliflozin reduces TNFa-induced reactive oxygen species through inhibition of the human endothelial Na+/H+ exchanger 1

Abstract Background Chronic low-grade inflammation, endothelial dysfunction and oxidative stress are major interactive pathways contributing to the pathogenesis of HFpEF. The sodium/glucose cotransporter 2 (SGLT2) inhibitor Empagliflozin (EMPA) inhibits inflammation-induced ROS generation in endothelial cells. EMPA also lowers Na+/H+ exchanger 1 (NHE1) activity and cytoplasmatic Na+ levels ([Na+]c) in cardiomyocytes. Purpose It is unknown how inflammation causes oxidative stress in endothelial cells and how EMPA can mitigate this. Here we examined for human endothelial cells whether 1) inflammatory mediators activate NHE1 activity, 2) increased [Na+]c mediates the inflammation-induced ROS production, and 3) EMPA reduces inflammation-induced ROS through NHE1 inhibition. Methods Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) were incubated with vehicle (V), 10ng/mL TNFα, 1μM EMPA or the canonical NHE1 inhibitor 10μM Cariporide. NHE1 activity was measured using the intracellular pH sensitive seminaphtharhodafluor fluorescence, by the pH recovery rate after an ammonium pulse. Intracellular ROS were detected by fluorescent live cell imaging in the presence of 10ng/mL TNFα or under increased Na+ conditions using 100nM ouabain (partial inhibition of the Na+/K+ pump). [Na+]c was measured using the sodium-binding benzofuran isophthalate 1 fluorescent probe. Results TNFα enhanced NHE1 activity in HCAECs (Fig a, in Δ[H+]/sec, V 0.81±0.14, TNFα 1.86±0.35 p&amp;lt;0.05). Increasing [Na+]c by ouabain elevated ROS generation (Fig b, in mM, HUVECs; V 5.6±1.3, ouabain 25.2±1.6 p&amp;lt;0.001, HCAECs; V 18.5±2.4, ouabain 91.2±32.6 p&amp;lt;0.05). EMPA reduced NHE1 activity in HUVECs (in Δ[H+]/sec, V 3.55±0.54, EMPA 1.66±0.25 p&amp;lt;0.01 vs V, Cariporide 0.95±0.11 p&amp;lt;0.001 vs V). HCAECs showed similar results. EMPA lowered [Na+]c (HUVECs; from 8.8±0.9 to 6.0±0.5 mM, p&amp;lt;0.05, HCAECs; from 7.8±0.7 to 4.0±0.8 mM, p&amp;lt;0.001). ROS generation was reversed in the presence of EMPA and/or Cariporide indicating that the effect was mediated by NHE inhibition (Fig c). Conclusions TNFα induces oxidative stress in EC through NHE1 activation and the consequently increased [Na+]c. EMPA acutely inhibits TNFα-induced ROS generation by inhibiting NHE1 and lowering [Na+]c in human cardiac and non-cardiac endothelial cells. Our data suggest that the previously reported cardiac effects of EMPA on NHE1 and [Na+]c are also present in endothelial cells. Endothelial ion homeostasis may be a valuable target to reduce inflammation-induced ROS generation and can constitute the pathway through which SGLT2 inhibitors offer protection in HFpEF patients, as examined in the EMPEROR-Preserved trial. Funding Acknowledgement Type of funding sources: None. Figure 1

Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett's Esophageal Cell Line.

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett’s esophagus, and this is presumably a compensatory mechanism against this toxic agent.

Mouse organoid culture is a suitable model to study esophageal ion transport mechanisms.

Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated three-dimensional (3D) esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250-300 µm and were generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl-/HCO3- anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR), and anoctamin 1 Cl- channels was detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.

Abstract P160: C1q/TNF-related Protein 1 Induces Obesity-related Hypertension In Mice Via Renal Na+ Retention In The Proximal Tubules

A recently identified adipokine C1q/TNF-related protein 1 (CTRP1) displayed a potent beneficial effect in managing metabolic disorders during type 2 diabetes and obesity. However, little is known about a potential role of CTRP1 in regulation of renal function and blood pressure (BP) in the context of obesity. Therefore, the present study aimed to determine the effect of a recombinant CTRP1 protein (termed CTRP1-His) on blood pressure in mice with diet-induced obesity (DIO). 8-mo-old male C57/B6 mice were fed a high-fat diet (60 Kcal% fat) for eight weeks and were infused with vehicle or CTRP1-His (10 ng/min/kg) via subcutaneously implanted osmotic minipump for the last seven days. BP parameters were recorded using telemetry devices. Consistent with other groups’ studies, mice infused with CTRP1-His exhibited remarkably improved multiple metabolic parameters, including hyperglycemia and hyperinsulinemia. Compared to the vehicle group, BP responses to CTRP1-His treatment developed a modest but significant increase in BP (MAP on day 7 [mmHg]: 122.3 ± 2.2 vs. 109 ± 1.73, n = 3 per each group, p &lt; 0.01) accompanied with decreased hematocrit (Hct [%]: 43.8 ± 1.3 vs. 49 ± 1.2 %, p &lt; 0.05), an index of plasma volume expansion. The DIO mice infused with CTRP1-His demonstrated a reduced urinary sodium excretion (U Na /Creatinine [mmol/mg]: 0.17 ± 0.04 vs. 0.27 ± 0.06, p = 0.07). By immunoblotting CTRP1-His infusion induced significant upregulation of renal abundances of p-NHE3, V2R, and AQP2 but suppressed obesity-induced renal abundances of p-NCC/NCC, p-NKCC2/NKCC2, and cleaved α-ENaC protein. Interestingly, CTRP1-His infusion tends to decrease albuminuria (urine albumin/Creatinine [mg/g]: 78.8 ± 7.7 vs. 120.5 ± 14, p = 0.07) in the DIO mice. Overall, our results indicated that CTRP1-His exerted a pressor effect in DIO mice via stimulating sodium-water retention through activation of NHE3 and AQP2 associated with compensatory attenuation of Na+ transporters in the distal nephron.

Glomerular Mesangial Cell pH Homeostasis Mediates Mineralocorticoid Receptor-Induced Cell Proliferation

Mineralocorticoids (e.g., aldosterone) support chronic inflammatory tissue damage, including glomerular mesangial injury leading to glomerulosclerosis. Furthermore, aldosterone leads to activation of the extracellular signal-regulated kinases (ERK1/2) in rat glomerular mesangial cells (GMC). Because ERK1/2 can affect cellular pH homeostasis via activation of Na+/H+-exchange (NHE) and the resulting cellular alkalinization may support proliferation, we tested the hypothesis that aldosterone affects pH homeostasis and thereby cell proliferation as well as collagen secretion also in primary rat GMC. Cytoplasmic pH and calcium were assessed by single-cell fluorescence ratio imaging, using the dyes BCECF or FURA2, respectively. Proliferation was determined by cell counting, thymidine incorporation and collagen secretion by collagenase-sensitive proline incorporation and ERK1/2-phosphorylation by Western blot. Nanomolar aldosterone induces a rapid cytosolic alkalinization which is prevented by NHE inhibition (10 µmol/L EIPA) and by blockade of the mineralocorticoid receptor (100 nmol/L spironolactone). pH changes were not affected by inhibition of HCO3− transporters and were not dependent on HCO3−. Aldosterone enhanced ERK1/2 phosphorylation and inhibition of ERK1/2-phosphorylation (10 µmol/L U0126) prevented aldosterone-induced alkalinization. Furthermore, aldosterone induced proliferation of GMC and collagen secretion, both of which were prevented by U0126 and EIPA. Cytosolic calcium was not involved in this aldosterone action. In conclusion, our data show that aldosterone can induce GMC proliferation via a MR and ERK1/2-mediated activation of NHE with subsequent cytosolic alkalinization. GMC proliferation leads to glomerular hypercellularity and dysfunction. This effect presents a possible mechanism contributing to mineralocorticoid receptor-induced pathogenesis of glomerular mesangial injury during chronic kidney disease.

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

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

Cardioprotection by SGLT2 Inhibitors-Does It All Come Down to Na+?

Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are emerging as a new treatment strategy for heart failure with reduced ejection fraction (HFrEF) and—depending on the wistfully awaited results of two clinical trials (DELIVER and EMPEROR-Preserved)—may be the first drug class to improve cardiovascular outcomes in patients suffering from heart failure with preserved ejection fraction (HFpEF). Proposed mechanisms of action of this class of drugs are diverse and include metabolic and hemodynamic effects as well as effects on inflammation, neurohumoral activation, and intracellular ion homeostasis. In this review we focus on the growing body of evidence for SGLT2i-mediated effects on cardiac intracellular Na+ as an upstream mechanism. Therefore, we will first give a short overview of physiological cardiomyocyte Na+ handling and its deterioration in heart failure. On this basis we discuss the salutary effects of SGLT2i on Na+ homeostasis by influencing NHE1 activity, late INa as well as CaMKII activity. Finally, we highlight the potential relevance of these effects for systolic and diastolic dysfunction as well as arrhythmogenesis.

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.

Empagliflozin Inhibits Proximal Tubule NHE3 Activity, Preserves GFR, and Restores Euvolemia in Nondiabetic Rats with Induced Heart Failure.

SGLT2 inhibitors reduce the risk of heart failure (HF) mortality and morbidity, regardless of the presence or absence of diabetes, but the mechanisms underlying this benefit remain unclear. Experiments with nondiabetic HF rats tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) inhibits proximal tubule (PT) NHE3 activity and improves renal salt and water handling. Male Wistar rats were subjected to myocardial infarction or sham operation. After 4 weeks, rats that developed HF and sham rats were treated with EMPA or untreated for an additional 4 weeks. Immunoblotting and quantitative RT-PCR evaluated SGLT2 and NHE3 expression. Stationary in vivo microperfusion measured PT NHE3 activity. EMPA-treated HF rats displayed lower serum B-type natriuretic peptide levels and lower right ventricle and lung weight to tibia length than untreated HF rats. Upon saline challenge, the diuretic and natriuretic responses of EMPA-treated HF rats were similar to those of sham rats and were higher than those of untreated HF rats. Additionally, EMPA treatment prevented GFR decline and renal atrophy in HF rats. PT NHE3 activity was higher in HF rats than in sham rats, whereas treatment with EMPA markedly reduced NHE3 activity. Unexpectedly, SGLT2 protein and mRNA abundance were upregulated in the PT of HF rats. Prevention of HF progression by EMPA is associated with reduced PT NHE3 activity, restoration of euvolemia, and preservation of renal mass. Moreover, dysregulation of PT SGLT2 may be involved in the pathophysiology of nondiabetic HF.

Abstract 1350: Regulation of pH by growth factors is dependent on the expression of the calcineurin homologous protein-2 in human bone sarcoma cells

Abstract Bone sarcoma is a malignant tumor that includes multiple subtypes. The osteosarcoma is the sixth most common malignancy in adolescents, while chondrosarcoma is primarily a disease of adults. Current treatments for both osteosarcoma and chondrosarcoma have not demonstrated improvement in patients' survival over the past three decades. Extracellular acidosis affects osteoblast and chondrocyte function by reducing bone mineralization and increasing production of cartilage matrix, respectively. Thereby, it weakens bone matrix and expands cartilage matrix, and may play a role in the development of bone characteristics seen in patients with osteosarcoma or chondrosarcoma. Development of extracellular acidosis in cancer is due to dysregulation of pH dynamics mediated by constitutive activation of Na+/H+ exchanger-1 (NHE1) activity, the main transporter responsible for pH regulation. Current therapeutic agents that target inhibition of NHE1 activity are ineffective due to broad inhibitory effects. The calcineurin homologous protein (CHP) family binds to and regulates NHE1 activity. CHP isoform 1 (CHP1) is expressed in non-cancerous cells and forms a dynamic complex with NHE1. On the other hand, CHP isoform 2 (CHP2) is expressed primarily in cancerous cells and has a stronger binding affinity to NHE1 compared with CHP1. We hypothesize that CHP2 binding to NHE1 in cancer cells induces the over-activity of NHE1, making the extracellular pH of cancer cells more acidic than non-cancerous cells. We measured CHP1 or CHP2 protein expression levels in human osteoblast (hFOB), osteosarcoma (143B), and chondrosarcoma (SW1353) cells and studied whether the levels of CHP2 protein expression affected NHE activity in these cell lines. We determined that: 1. hFOB, 143B, and SW1353 cells expressed mainly NHE1; NHE activity measured using spectrofluorometry was completely blocked by a highly selective NHE1 inhibitor (zoniporide, 10-8 M); 2. NHE1 activity was increased by serum-nutrient deprivation, used to mimic a cancerous microenvironment and NHE1 over-activity was associated with increased levels of CHP2 protein expression; 3. silencing of CHP2 by shRNA inhibited NHE1 activity in response to serum-nutrient deprivation in 143B or SW1353 cells and restored the NHE1 response inducted by serum-deprivation in non-cancerous cells. Regulation of NHE1 through CHP2 is proposed as a promising novel target in sarcoma treatment and to overcome the wide range of side effects induced by inhibitors of NHE1 activity. Citation Format: Tiffany Chang, Serena S. Luong, Adam P. Zobel, Elitsa Ananieva, Victor Babich, Francesca Di Sole. Regulation of pH by growth factors is dependent on the expression of the calcineurin homologous protein-2 in human bone sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1350.

Ethyl isopropyl amiloride decreases oxidative phosphorylation and increases mitochondrial fusion in clonal untransformed and cancer cells.

Many cancer cells, regardless of their tissue origin or genetic landscape, have increased expression or activity of the plasma membrane Na-H exchanger NHE1 and a higher intracellular pH (pHi) compared with untransformed cells. A current perspective that remains to be validated is that increased NHE1 activity and pHi enable a Warburg-like metabolic reprogramming of increased glycolysis and decreased mitochondrial oxidative phosphorylation. We tested this perspective and find it is not accurate for clonal pancreatic and breast cancer cells. Using the pharmacological reagent ethyl isopropyl amiloride (EIPA) to inhibit NHE1 activity and decrease pHi, we observe no change in glycolysis, as indicated by secreted lactate and intracellular pyruvate, despite confirming increased activity of the glycolytic enzyme phosphofructokinase-1 at higher pH. Also, in contrast to predictions, we find a significant decrease in oxidative phosphorylation with EIPA, as indicated by oxygen consumption rate (OCR). Decreased OCR with EIPA is not associated with changes in pathways that fuel oxidative phosphorylation or with mitochondrial membrane potential but occurs with a change in mitochondrial dynamics that includes a significant increase in elongated mitochondrial networks, suggesting increased fusion. These findings conflict with current paradigms on increased pHi inhibiting oxidative phosphorylation and increased oxidative phosphorylation being associated with mitochondrial fusion. Moreover, these findings raise questions on the suggested use of EIPA-like compounds to limit metabolic reprogramming in cancer cells.

Unique Regulation of Coupled NaCl Absorption by Inducible Nitric Oxide in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Intestinal Inflammation.

In the small intestine, Na:H (NHE3) and Cl:HCO3 (DRA or PAT1) exchangers present in the brush border membrane (BBM) of absorptive villus cells are primarily responsible for the coupled absorption of NaCl, the malabsorption of which causes diarrhea, a common symptom of inflammatory bowel disease (IBD). Inducible nitric oxide (iNO), a known mediator of inflammation, is increased in the mucosa of the chronically inflamed IBD intestine. An SAMP1/YitFc (SAMP1) mouse, a spontaneous model of chronic ileitis very similar to human IBD, was used to study alterations in NaCl absorption. The SAMP1 and control AKR mice were treated with I-N(6)-(1-Iminoethyl)-lysine (L-NIL) to inhibit iNO production, and DRA/PAT1 and NHE3 activities and protein expression were studied. Though Na:H exchange activity was unaffected, Cl:HCO3 activity was significantly decreased in SAMP1 mice due to a reduction in its affinity for Cl, which was reversed by L-NIL treatment. Though DRA and PAT1 expressions were unchanged in all experimental conditions, phosphorylation studies indicated that DRA, not PAT1, is affected in SAMP1. Moreover, the altered phosphorylation levels of DRA was restored by L-NIL treatment. Inducible NO mediates the inhibition of coupled NaCl absorption by decreasing Cl:HCO3 but not Na:H exchange. Specifically, Cl:HCO3 exchanger DRA but not PAT1 is regulated at the level of its phosphorylation by iNO in the chronically inflamed intestine.

Acute and chronic temperature dependence of Na+/H+ exchange activity of Pimephales promelas gills

Na+/H+ exchangers (NHE) mediate at least part of Na+ entry into gill epithelia via Na+/NH4+ exchange. For homeostasis, Na+ entry into and exit via Na+/K+ ATPase from gill epithelia must balance. Na+/K+ ATPase activity is reduced in cold- compared to warm-acclimated freshwater temperate fish. We hypothesized gill NHE activity is greater in warm- than cold-acclimated fish when measured at acclimation temperatures, and NHE activity displays a temperature dependence similar to Na+/K+ ATPase. Since NHE mRNA expression does not differ, we measured the Na+-dependence of pH-induced Na+ fluxes in gill vesicles from warm- and cold-acclimated fathead minnows at 20o and 7 °C, and calculated maximum transport rates (Vmax) and Na+ K1/2s. We also measured NH4+-induced Na+ fluxes and Na+-induced H+ fluxes. In vesicles from warm-acclimated fish, NHE Vmaxs were 278 ± 33 and 149 ± 23 arbitrary unit/s (au/s) and Na+ K1/2s were 12 ± 4 and 6 ± 4 mmol/l when assayed at 20o and 7 °C (p < 0.004), respectively. In vesicles from cold-acclimated fish, Vmaxs were 288 ± 35 and 141 ± 13 au/s and Na+ K1/2s 17 ± 5 and 7 ± 2 mmol/l when assayed at 20o and 7 °C (p < 0.002), respectively. Na+-induced H+ fluxes were 98 ± 8 and 104 ± 26 au/s in warm- and cold-acclimated fish assayed at 20 °C, respectively. Na+/NH4+ exchange was 120 ± 11 and 158 ± 13 au/s in warm- and cold-acclimated fish, respectively. Conclusions: Gill NHE activity was greater in warm- than cold-acclimated fish assayed at acclimation temperatures. The temperature dependence of NHE activity was similar in both groups, but differed from that reported for Na+/K+ ATPase suggesting complex mechanisms to maintain Na+ homeostasis.

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.