NHE3 Expression Research Articles

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.

The Role of the Androgen Receptor in Sexual Dimorphisms of Renal Ammonia Metabolism

Female mice excrete ~2x more ammonia than do male mice, and this appears to result from sex-differences in ammoniagenic enzyme expression in the proximal tubule (PT) and differences in ammonia transporter expression in the PT, TAL and collecting duct. Orchiectomy studies, with and without testosterone replacement, showed that many, but not all, of these sex-differences were mediated by testosterone. We showed recently that the androgen receptor (AR) is present in the PT in both male and female kidney, and not detectably in other renal epithelial cells. This study's objective was to determine AR's role in sexual dimorphisms in ammonia handling. To avoid known systemic effects of AR blockade/deletion, we generated mice with kidney-specific AR deletion (KS-AR-KO) using Cre/loxP techniques (AR floxed mice and Pax8-Cre mice); control (C) mice were Pax8-Cre-negative littermates. Plasma testosterone did not differ significantly between KO and C mice in either sex (M-C, 67±74 ng/dl; M-KO, 239±471; F-C, 27±10; F-KO, 29±11; P=NS; N= 8-11 in each group). In male mice, KS-AR-KO increased ammonia excretion significantly (M-C, 44±14 µmol/day; M-KO, 92±74; P < 0.05; N= 8-11 in each group); this occurred despite no significant difference in food intake, a major determinant of endogenous acid production; there was no significant difference in female mice. KS-AR-KO did not alter serum Na+, K+, or HCO3- significantly in either sex. Expression of phosphoenolpyruvate carboxykinase (PEPCK), a major proximal tubule (PT) ammonia generating protein, and NKCC2, the major mechanism of TAL ammonia reabsorption, were increased significantly by KS-AR-KO in male mice, but not in female mice. KS-AR-KO decreased expression of NHE-3, the major mechanism of PT ammonia secretion, and NBCe1-A, a basolateral PT transporter that regulates PT ammonia metabolism, in male mice, but not in female mice, and did not alter the sex-specific difference in collecting duct Rhbg and Rhcg expression in either sex. Renal structural analysis showed AR deletion decreased kidney size (M-C, 222±27 mg; M-KO, 175±23; P<0.05; N= 5-6 in each group) and cortical proximal tubule volume density (C, 61±2%; KO, 42±2; P<0.05; N= 5-6 in each group) in male mice, but did not alter either parameter in females. KS-AR-KO did not alter the sexual dimorphism of collecting duct volume density in either sex. We conclude that sex-differences in ammonia generation and excretion involve AR-dependent signaling pathways in male, but not female, kidney that lead to increased PT PEPCK and TAL NKCC2 expression. Adaptive responses in NHE-3 and NBCe1-A limit the magnitude of the effect on ammonia excretion, and the sex differences in the collecting duct are not mediated by AR. Since AR is not present in the TAL, the effect of KS-AR-KO on male NKCC2 expression likely involves PT-dependent paracrine signaling. Finally, the greater kidney size and PT volume density in male mice is the result of PT AR signaling.

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.

Lean Mice Show Sex‐Specific Effects of Early Life Stress on Water and Electrolyte Homeostasis and Renal Function

Early life stress (ELS) is associated with increases in body mass index and systolic blood pressure in adult life, leading to obesity and obesity-induced hypertension, major risk factors for cardiovascular disease (CVD). Previously, we have shown that Maternal Separation and Early Weaning (MSEW), a model that mimics the effects of ELS observed in humans, exacerbates hypertensive stimuli responses in male and female mice. The aim of this study was to investigate the status of the water and electrolyte homeostasis (WEH) at baseline in mice exposed to MSEW. Therefore, we determined water intake and excretion, plasma and urinary electrolytes, glomerular filtration rate (GFR) and sodium transporters expression along the nephron in lean mice. MSEW litter pups were separated from dams from postnatal day (PND) 2 to 16, followed by early weaning on PND 17. Litters that remained undisturbed and were weaned on PND 21 served as controls. At weaning, mice were placed on a low-fat diet (LF, 10% Kcal from fat) for 12 weeks. Single mouse metabolic cages were used to measure water intake and urine excretion. GFR was measured using a transcutaneous technique. MSEW males fed a LF showed an increase in water intake compared to controls (4.14 ± 0.32 vs. 3.4 ± 0.36 mL/day respectively, P<0.05), urine excretion (1.01 ± 0.16 vs. 0.61 ± 0.07 mL/day, P<0.05), and potassium excretion (12.05 ± 2.48 vs. 7.29 ± 1.49 µmol/day/g BW, P<0.05). MSEW mice showed no differences in plasma sodium (165.16 ± 2.44 vs. 161.34 ± 2.67 mmol/L), plasma potassium (5.42 ± 0.19 vs. 5.86 ± 0.16 mmol/L) or sodium excretion compared to controls (3.65 ± 0.72 vs. 3.24 ± 1.43 µmol/day/g BW, respectively). In addition, MSEW males showed increased GFR compared to controls (1.12 ± 0.05 vs. 0.90 ± 0.03 mL/min/100g BW, P<0.05). Renal cortex NHE3 expression was increased in MSEW compared to control mice (7.74 ± 2.22 vs. 1.2 ± 0.25 2^ddCt, respectively, P<0.05) while NCC expression was decreased compared to controls (0.38 ± 0.13 vs. 1.25 ± 0.32 2^ddCt respectively, P<0.05). Yet, no significant changes between MSEW and control mice were found in renal transporters NKCC2 (0.91 ± 0.26 vs. 1.23 ± 0.29 2^ddCt, respectively), or in the ENaC subunits ENaCα (0.97 ± 0.09 vs. 1.12 ± 0.21 2^ddCt), ENaCβ (1.14 ± 0.13 vs. 1.14 ± 0.27 2^ddCt), and ENaCγ (0.88 ± 0.13 vs. 1.08 ± 0.17 2^ddCt). Lean female MSEW mice showed no significant differences in water intake, urine excretion, urinary potassium, GFR, or renal transporters expression. Both increased NHE3 and reduced NCC expression have been linked to a greater potassium waste. These results show that the MSEW effects on WEH in lean mice are sex-specific, where only male mice display hyperfiltration and alterations in specific renal transporters expression. Chronic hyperfiltration may result in kidney damage and over time, chronic kidney disease. This could be aggravated in animals exposed to a secondary stressor such as a high-fat or high-salt diet. Ongoing studies of plasma and urine osmolarity may provide insights into the potential mechanisms by which MSEW male mice show hyperfiltration in lean healthy and normotensive mice.

Exchange Protein Directly Activated by cAMP Isoform 2 (Epac2) but Not Isoform 1 (Epac1) Is Critical for Renal Acid‐Base Handling

Cyclic adenosine monophosphate (cAMP) is a critical second messenger of numerous systemic and local signals affecting water-electrolyte transport and pH regulation by the renal tubule. Exchange proteins directly activated by cAMP (Epacs) are guanine nucleotide exchange factors commonly operating independently from the classical cAMP-protein kinase A (PKA) signaling. We previously demonstrated that both Epac isoforms, namely Epac1 and Epac2 are functionally expressed in the proximal tubule and the collecting duct. Epac1 -/- and Epac2 -/- mice exhibit urinary concentrating defect and salt wasting in response to hypovolemia. Since the proximal tubule and the collecting duct are central for urinary acid/base excretion, we examined the role of Epac1 and Epac2 in maintaining systemic pH balance. EpacWT, Epac1-/-, and Epac2 -/- mice had comparable arterial pH of 7.33±0.02, 7.33±0.04, and 7.30±0.02 respectively at the baseline. However, urinary pH was significantly lower only in Epac2 -/- (5.96±0.06), but not Epac1 -/- (6.61±0.15) when compared to EpacWT (6.90±0.38). Acid loading with NH4Cl supplementation in water for 3 days mildly reduced arterial pH in EpacWT and Epac1-/- to 7.27±0.01 and 7.24±0.03 respectively, but induced significantly greater acidosis (7.12±0.03) in Epac2 -/- mice. This was also associated with significantly lower blood HCO3- levels in Epac2 -/- compared to Epac1-/- and EpacWT: 18±1 mM/l, 23±2 mM/l, and 23±1 mM/l, respectively. Furthermore, NH4Cl supplementation led to marked reduction in urinary pH levels of EpacWT and Epac1-/-, but failed to significantly decrease already low urinary pH in Epac2 -/- (5.86±0.03). While NHE-3 levels in the proximal tubule were significantly reduced in Epac1-/-mice (~50%), this was more pronounced in Epac2-/- mice (~90%). Furthermore, pHi analysis in freshly isolated split-opened collecting ducts revealed altered recovery after acidification in intercalated cells of Epac2-/- mice. Overall, we report that Epac2 plays a critical role in renal adaptation to acidification. Epac2 deficiency compromises urinary acid excretion by reducing NHE-3 expression in the proximal tubule and distorting titratable acid excretion by the collecting duct intercalated cells leading to severe acidosis. In contrast, Epac1 does not play a major role in renal acid/base handling. The observed reduction in NHE-3 levels in Epac1-/- are effectively compensated by the acid excretion in the collecting duct and thus does not lead to prominent alteration in blood pH. We speculate that pharmacology-based manipulation with Epac2, but not Epac1 might be of relevance in managing acid-base disorders in the clinical setting.

Dihydroartemisinin prompts amplification of photodynamic therapy-induced reactive oxygen species to exhaust Na/H exchanger 1-mediated glioma cells invasion and migration

Photodynamic therapy (PDT) is a promising glioma therapy; however, its efficacy is compromised due to the PDT-induced reactive oxygen species (ROS) production being limited by the local hypoxic tumor microenvironment. Furthermore, Hypoxia activates sodium/hydrogen exchanger 1 (NHE1), an essential component for tumor progression and metastasis, enables glioma cells (GC) to escape PDT-mediated phototoxicity via increased H+ extrusion. However, interactions between NHE1 expression with ROS level involving response of GC remain unclear. Dihydroartemisinin (DHA), a ROS generator, has extensive anti-tumor effects. This study aimed to explore whether PDT along with DHA could amplify the total ROS levels and diminish GC invasion and migration by inhibiting NHE1 expression. Proliferation and invasion of U251 and LN229 cells were evaluated under different treatments using cell counting Kit-8 (CCK-8), transwell, and wound healing assays. ROS levels were measured using fluorescence probes and flow cytometry. NHE1 levels were detected by immunofluorescence and western blotting. Co-treatment effects and molecular events were further confirmed in a bilateral tumor-bearing nude mouse model. PDT with synergistic DHA significantly increased the total abundance of ROS to further suppress the invasion and migration of GC by reducing NHE1 levels in vitro. Using a bilateral glioma xenograft mouse model with primary and recurrent gliomas, we found that PDT markedly suppressed primary tumor growth, while PDT in synergy with DHA also suppressed recurrent tumors, and improved overall survival by regulating the ROS-NHE1 axis. No evident side effects were observed. Our results suggest that PDT with DHA can amplify the total ROS levels to weaken GC invasion and migration by suppressing NHE1 expression in vitro and in vivo, thus abolishing the resistance of GC to PDT. The synergistic therapy of PDT and DHA therefore represents a more efficient and safe strategy for comprehensive glioma treatment.

Long-Term Angiotensin II Infusion Induces Oxidative and Endoplasmic Reticulum Stress and Modulates Na+ Transporters Through the Nephron.

High plasma angiotensin II (Ang II) levels are related to many diseases, including hypertension, and chronic kidney diseases (CKDs). Here, we investigated the relationship among prolonged Ang II infusion/AT1 receptor (AT1R) activation, oxidative stress, and endoplasmic reticulum (ER) stress in kidney tissue. In addition, we explored the chronic effects of Ang II on tubular Na+ transport mechanisms. Male Wistar rats were subjected to sham surgery as a control or prolonged Ang II treatment (200 ng⋅kg–1⋅min–1, 42 days) with or without losartan (10 mg⋅kg–1⋅day–1) for 14 days. Ang II/AT1R induced hypertension with a systolic blood pressure of 173.0 ± 20 mmHg (mmHg, n = 9) compared with 108.0 ± 7 mmHg (mmHg, n = 7) in sham animals. Under these conditions, gene and protein expression levels were evaluated. Prolonged Ang II administration/AT1R activation induced oxidative stress and ER stress with increased Nox2, Nox4, Cyba and Ncf1 mRNA expression, phosphorylated PERK and eIF2α protein expression as well as Atf4 mRNA expression. Ang II/AT1R also raised Il1b, Nfkb1 and Acta2 mRNA expression, suggesting proinflammatory, and profibrotic effects. Regarding Na+ tubular handling, Ang II/AT1R enhanced cortical non-phosphorylated and phospho/S552/NHE3, NHE1, ENaC β, NKCC2, and NCC protein expression. Our results also highlight the therapeutic potential of losartan, which goes beyond the antihypertensive effect, playing an important role in kidney tissue. This treatment reduced oxidative stress and ER stress signals and recovered relevant parameters of the maintenance of renal function, preventing the progression of Ang II-induced CKD.

Sex hormones regulate NHE1 functional expression and brain endothelial proteome to control paracellular integrity of the blood endothelial barrier

BackgroundSex hormones have been implicated in pH regulation of numerous physiological systems. One consistent factor of these studies is the sodium-hydrogen exchanger 1 (NHE1). NHE1 has been associated with pH homeostasis at epithelial barriers. Hormone fluctuations have been implicated in protection and risk for breaches in blood brain barrier (BBB)/blood endothelial barrier (BEB) integrity. Few studies, however, have investigated BBB/BEB integrity in neurological disorders in the context of sex-hormone regulation of pH homeostasis. Methods//ResultsPhysiologically relevant concentrations of 17-β-estradiol (E2, 294 pM), progesterone (P, 100 nM), and testosterone (T,3.12 nM) were independently applied to cultured immortalized bEnd.3 brain endothelial cells to study the BEB. Individual gonadal hormones showed preferential effects on extracellular pH (E2), 14C-sucrose uptake (T), stimulated paracellular breaches (P) with dependence on functional NHE1 expression without impacting transendothelial resistance (TEER) or total protein expression. While total NHE1 expression was not changed as determined via whole cell lysate and subcellular fractionation experiment, biotinylation of NHE1 for surface membrane expression showed E2 reduced functional expression. Quantitative proteomic analysis revealed divergent effects of 17-β-estradiol and testosterone on changes in protein abundance in bEnd.3 endothelial cells as compared to untreated controls. ConclusionsThese data suggest that circulating levels of sex hormones may independently control BEB integrity by 1) regulating pH homeostasis through NHE1 functional expression and 2) modifying the endothelial proteome.

Thioredoxin 1 (TRX1) Overexpression Cancels the Slow Force Response (SFR) Development.

The stretch of cardiac muscle increases developed force in two phases. The first phase occurs immediately after stretch and is the expression of the Frank–Starling mechanism, while the second one or slow force response (SFR) occurs gradually and is due to an increase in the calcium transient amplitude. An important step in the chain of events leading to the SFR generation is the increased production of reactive oxygen species (ROS) leading to redox sensitive ERK1/2, p90RSK, and NHE1 phosphorylation/activation. Conversely, suppression of ROS production blunts the SFR. The purpose of this study was to explore whether overexpression of the ubiquitously expressed antioxidant molecule thioredoxin-1 (TRX1) affects the SFR development and NHE1 phosphorylation. We did not detect any change in basal phopho-ERK1/2, phopho-p90RSK, and NHE1 expression in mice with TRX1 overexpression compared to wild type (WT). Isolated papillary muscles from WT or TRX1-overexpressing mice were stretched from 92 to 98% of its maximal length. A prominent SFR was observed in WT mice that was completely canceled in TRX1 animals. Interestingly, myocardial stretch induced a significant increase in NHE1 phosphorylation in WT mice that was not detected in TRX1-overexpressing mice. These novel results suggest that magnification of cardiac antioxidant defense power by overexpression of TRX1 precludes NHE1 phosphorylation/activation after stretch, consequently blunting the SFR development.

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.

Shikonin suppresses the epithelial-to-mesenchymal transition by downregulating NHE1 in bladder cancer cells.

Shikonin (SK) is the major bioactive component extracted from the roots of Lithospermum erythrorhizon with anticancer activity. SK could inhibit the epithelial-to-mesenchymal transition (EMT) of cancer cells. However, the underlying mechanism is elusive. In the present study, the inhibitory activities of SK on proliferation, invasion and migration were examined in bladder cancer (BC) cells. SK potently decreased the viabilities of BC cells but showed less cytotoxicity to normal bladder epithelial cells. Moreover, SK reversed the EMT, suppressed the migration and invasion of BC cells. Intriguingly, NHE1, the major proton efflux pump, was dramatically down-regulated by SK. The EMT-inhibitory effect of SK was mediated by NHE1 down-regulation, as NHE1-overexpress alleviated while Cariporide (NHE1 inhibitor) enhanced this effect. Further, enforced alkalinization of intracellular pH (pHi) reversed the EMT-inhibitory effect of SK, indicating a key role of acidic pHi in this process. Finally, elevated NHE1 expression was observed in human bladder cancer tissues. Collectively, this research reveals a supportive effect of NHE1 and alkaline pHi on EMT. SK can suppress EMT through inhibiting NHE1 and hence inducing an acidic pHi.

Chemical composition and anti-inflammatory activity of n-butanol extract of Piper sarmentosum Roxb. In the intestinal porcine epithelial cells (IPEC-J2)

Ethnopharmacological relevancePiper sarmentosum Roxb. (PS) is a terrestrial herb primarily distributed in tropical and subtropical regions of Asia. It is widely used in folk medicine in certain countries of Southeast Asia for the treatment of fever, toothache, coughing and pleurisy, which showed the anti-inflammatory activity of PS. Aim of the studyThis study aimed to investigate the chemical constituents and the molecular mechanism and related metabolic pathway by which n-butanol extract of PS (PSE-NB) exerts its anti-inflammatory effects. Materials and methodsChemical constituents of PSE-NB was analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique. Anti-inflammatory effects of PSE-NB were investigated in lipopolysaccharide (LPS)-induced IPEC-J2 cells. ResultsIn total, 218 compounds, including 94 alkaloids and 26 phenolics were tentatively identified, which indicating alkaloids and phenolics were the main constituents of PSE-NB. In addition, the current cell experiment in vitro showed that PSE-NB (10–500 μg/mL) pre-treatment before LPS stimulation significantly decreased mRNA expression of IL-1β, IL-6 and TNF-α in IPEC-J2 cells compared with LPS treatment (p < 0.05). PSE-NB improved mRNA expression of tight junction proteins (ZO-1 and Occludin) and NHE3, which were reduced by LPS stimulation (p < 0.05). Moreover, PSE-NB (10 μg/mL) alleviated LPS-induced protein expression of p65 and p-p65 (p < 0.05), and reduced p65 translocation into the nucleus induced by LPS. At the same time, metabolic pathway analysis indicated that PSE-NB exerts anti-inflammatory effects mainly via augmentation of methionine metabolism in IPEC-J2 cells. ConclusionsTaken together, the results suggested that alkaloids and phenolics were the main constituents in PSE-NB. PSE-NB might attenuate LPS-induced inflammatory responses in IPEC-J2 cells by regulating NF-κB signaling pathway and intracellular metabolic pattern.

Decreased GLUT1/NHE1 RNA expression in whole blood predicts disease severity in patients with COVID-19.

AimsWe aimed to assess whether expression of whole‐blood RNA of sodium proton exchanger 1 (NHE1) and glucose transporter 1 (GLUT1) is associated with COVID‐19 infection and outcome in patients presenting to the emergency department with respiratory infections. Furthermore, we investigated NHE1 and GLUT1 expression in the myocardium of deceased COVID‐19 patients.Methods and resultsWhole‐blood quantitative assessment of NHE1 and GLUT1 RNA was performed using quantitative PCR in patients with respiratory infection upon first contact in the emergency department and subsequently stratified by SARS‐CoV‐2 infection status. Assessment of NHE1 and GLUT1 RNA using PCR was also performed in left ventricular myocardium of deceased COVID‐19 patients.NHE1 expression is up‐regulated in whole blood of patients with COVID‐19 compared with other respiratory infections at first medical contact in the emergency department (control: 0.0021 ± 0.0002, COVID‐19: 0.0031 ± 0.0003, P = 0.01). The ratio of GLUT1 to NHE1 is significantly decreased in the blood of COVID‐19 patients who are subsequently intubated and/or die (severe disease) compared with patients with moderate disease (moderate disease: 0.497 ± 0.083 vs. severe disease: 0.294 ± 0.0336, P = 0.036). This ratio is even further decreased in the myocardium of patients who deceased from COVID‐19 in comparison with the myocardium of non‐infected donors.ConclusionsNHE1 and GLUT1 may be critically involved in the disease progression of SARS‐CoV‐2 infection. We show here that SARS‐CoV‐2 infection critically disturbs ion channel expression in the heart. A decreased ratio of GLUT1/NHE1 could potentially serve as a biomarker for disease severity in patients with COVID‐19.

Abstract 16216: Increases in Renal Nkcc2 and Ncc Preceded the Elevation of Blood Pressure in Mice Treated With Clozapine

One of major side effects of clozapine, a common antipsychotic drug, is hypertension. In order to explore the pathogenesis of the drug-induced hypertension, we examined the renal sodium transport proteins and BP in the C57Bl6/J male mice treated with clozapine at different doses for 1 week. Clozapine at 20mg/kg/day via osmotic mini-pump increased SBP ( 110±0.6,mmHg, femoral artery, under anesthesia) and DBP (76.7±1) relative to vehicle (97.6±1.2/71.2±2.7) while the renal protein expression of NKCC2 (180±21, % of vehicle) and NCC (171±22), and ENaC-α (140±7), β(166±21) and γ(130±7) was increased without changes in NHE3 and α1-NKA (n=5/group). Clozapine at lower doses (5&amp;10mg/kg/day,IP) did not change the conscious SBP and DBP monitored daily by tail-cuff, even on day 7 in 5 mg/kg group (115.61±1.6/90.1±1.3) and in 10mg/kg group (116.8±2/89.0±1.6) relative to vehicle (111.9±1.5/86.9±1.4) (n=4/group). Urinary excretion of Na + &amp; K + and serum concentrations of Na + , K + , Cl - , creatinine were similar in all groups. However, renal protein expression of NKCC2 was increased in 5mg/kg (360±80, % of vehicle) and 10mg/kg (247±50) groups; NCC was increased at 5 mg/kg (175±9); α1-NKA was increased in 5mg/kg (216±5) and 10mg/kg (163±2) groups. No increases were found in the protein expression of NHE3 and ENaCs. Those increases in renal NKCC2 &amp;NCC at 5 &amp; 10 mg/kg were consistent with those at 20 mg/kg. AT1R was increased at 5 (221±20) &amp;10mg/kg (255±21) while renin (186±15) and ACE1 (186±5), not ACE2, were increased at 5 mg/kg suggesting an activation of RAAS in kidney. NOX4, not NOX2, was increased at 5 mg/kg (453±69) while NOS3, not NOS1&amp;2, was decreased at 5 (60±21) and 10mg/kg (64±5) groups. TNFα, not IL-6 was increased at 5 (222±18) &amp;10 mg/kg (321±26). In cultured mouse distal convoluted tubule cells, clozapine also increased NCC and α1-NKA at 1nM (169±4;156±7) and 10nm(175±23;183±15) respectively ( 24h,n=4/group). Those changes in kidney, including increases in sodium transport proteins, RAAS components, ROS generation enzymes, inflammation factors and decrease in NO synthase, preceded the elevation of BP suggesting that direct or indirect regulations of clozapine on those proteins may play an important role in the pathogenesis of the drug-induced hypertension.

Ubiquitin-specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity.

Na+ /H+ exchanger NHE3 of human or primates differs from NHE3 of other animals by having a PY motif, which mediates interaction with the E3 ubiquitin (Ub) ligase Nedd4-2. Ub-conjugation of human NHE3 by Nedd4-2 regulates endocytosis of NHE3 but does not affect its cellular expression. Because Ub-conjugation is a reversible process, the aim of this study is to identify deubiquitinating enzyme (DUB) regulating the post-endosomal fate of human NHE3. Using an activity-based chemical screening, we identified ubiquitin specific protease-7 (USP7) and USP10 that bind NHE3. The roles of DUBs in regulation of NHE3 were studied by determining the effects of silencing of USP7 and USP10. Knockdown of USP7 or USP10 resulted in increased NHE3 ubiquitination and decreased NHE3 expression at the surface membrane and cellular level. The endocytic retrieval of NHE3 was promoted by depletion of USP7 or USP10, with increased association of NHE3 with Rab5a and Rab7. Inhibition of USP7 and USP10 by chemical inhibitors or knockdown had an additive effect on NHE3. In addition, NHE3 half-life was reduced accounting for decreased NHE3 protein abundance. NHE3 is inhibited by protein kinase A. Activation of PKA by forskolin decreased the binding of USP7 and USP10 to NHE3, while increasing ubiquitination of NHE3. Knockdown of USP10 had an additive effect on PKA-dependent inhibition of NHE3. These findings demonstrate that USP7 and USP10 are DUBs that regulate NHE3 ubiquitination and expression, and reveal a new mechanism of NHE3 inhibition involving DUBs.

Arsenic trioxide-increased MDCK cells proliferation requires activator protein 1-mediated increase of the sodium/proton exchanger 1 activity

The release of protons (H+) occurs via the Na+/H+ exchanger isoform 1 (NHE1) leading to a stable intracellular pH (pHi) in MDCK cells. Chronic intake of arsenic trioxide (ATO), in the drinking water, associated with higher morbidity and mortality in neoplastic tissues. ATO increased NHE1 expression and activity, resulting in intracellular alkalization and higher MDCK cells proliferation. Since the pro-proliferative transcription factor activator protein 1 (AP-1) gets activated by al alkaline intracellular pH, a phenomenon paralleled by higher NHEs activity, we asked whether ATO-increased MDCK cells proliferation involves AP-1–dependent NHE1 activation. Cells were exposed (48 h) to ATO (0.05 μmol/L), SR11302 (1 μmol/L, AP-1 inhibitor), HOE-694 (100 nmol/L, NHE1 inhibitor) and EIPA (50 μmol/L, NHE1/NHE3 inhibitor) in the presence of S3226 (10 μmol/L, NHE3 inhibitor), concanamycin A (0.1 μmol/L, V-ATPases inhibitor), and Schering (10 μmol/L, H+/K+-ATPase inhibitor). [3H]Thymidine incorporation, cell counting, wound healing assay, and AP-1 activity were determined. The pHi was measured in cells pre-loaded (10 min) with 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (12 mmol/L) and exposed to NH4Cl (20 mmol/L). Basal pHi and recovery rate (dpHi/dt), intracellular buffer capacity (βi) and H+ flux (JH+) were determined. NHE1 protein abundance was measured by Western blotting and immunofluorescence. ATO increased the cell growth (1.5 fold), basal pHi (0.4 pHi units), dpHi/dt (1.8 fold), JH+ (1.4 fold), AP-1 activity and NHE1 protein abundance (1.3 fold). ATO also increased (1.5 fold) the nuclear/perinuclear NHE1 immunosignal. SR11302 and HOE-694 blocked ATO effects. Thus, ATO-increased proliferation resulted from AP-1–dependent NHE1 activation in MDCK cells.

Empagliflozin inhibits Na+ /H+ exchanger activity in human atrial cardiomyocytes.

AimsRecent clinical trials have proven gliflozins to be cardioprotective in diabetic and non‐diabetic patients. However, the underlying mechanisms are incompletely understood. A potential inhibition of cardiac Na+/H+ exchanger 1 (NHE1) has been suggested in animal models. We investigated the effect of empagliflozin on NHE1 activity in human atrial cardiomyocytes.Methods and resultsExpression of NHE1 was assessed in human atrial and ventricular tissue via western blotting. NHE activity was measured as the maximal slope of pH recovery after NH4 + pulse in isolated carboxy‐seminaphtarhodafluor 1 (SNARF1)‐acetoxymethylester‐loaded murine ventricular and human atrial cardiomyocytes. NHE1 is abundantly expressed in human atrial and ventricular tissue. Interestingly, compared with patients without heart failure (HF), atrial NHE1 expression was significantly increased in patients with HF with preserved ejection fraction and atrial fibrillation. The largest increase in atrial and ventricular NHE1 expression, however, was observed in patients with end‐stage HF undergoing heart transplantation. Importantly, acute exposure to empagliflozin (1 μmol/L, 10 min) significantly inhibited NHE activity to a similar extent in human atrial myocytes and mouse ventricular myocytes. This inhibition was also achieved by incubation with the well‐described selective NHE inhibitor cariporide (10 μmol/L, 10 min).ConclusionsThis is the first study systematically analysing NHE1 expression in human atrial and ventricular myocardium of HF patients. We show that empagliflozin inhibits NHE in human cardiomyocytes. The extent of NHE inhibition was comparable with cariporide and may potentially contribute to the improved outcome of patients in clinical trials.

Abstract 11: Role Of Pro-saas Peptides In Salt Resistance

The recently deorphanized G protein couple receptor 83 (GPR83) is expressed in proximal and distal convoluted tubules of the mouse kidney and in human renal proximal tubule cells (hRPTCs). Peptides derived from the cleavage of the precursor neuroendocrine hormone pro-SAAS (pro serine, alanine, alanine, serine), in particular PEN (proline, glutamic acid, and asparagine), have been proposed as the endogenous ligands of GPR83 but their function in the kidney is unknown. GPR83 is constitutively active; silencing (siRNA, 20 nM, 72h) its expression in hRPTCs decreases the phosphorylation of ERK1/2, p38, p90RSK and AKT (43%, 52%, 65%, and 25% respectively, n=4/group, P&lt;0.05 vs non-silencing siRNA). Conversely, PEN (proline, glutamic acid, and asparagine) treatment of hRPTCs (1 μM, 1h) increases ERK1/2, p38, p90RSK, and AKT phosphorylation (15-fold, 1.7-fold, 2-fold, 18-fold respectively, n=4/group, P&lt;0.05 vs vehicle). We hypothesized that GPR83 and its ligands PEN and pro-SAAS are involved in the regulation of blood pressure. Global deletion or renal-specific silencing of the Gpr83 gene in mice increased systolic blood pressure (SBP). High salt diet increased Gpr83 transcription 2-fold (P&lt;0.05; n=4/group) in SJL/J and BALB/c salt-resistant mice, relative to C57Bl/6J salt-sensitive mice. Renal-restricted infusion of PEN (1 μg/day; 7 days) did not affect SBP, slightly decreased Na + excretion and renal NHE3 expression (P&lt;0.05 all) and did not affect renal Na + /K + -ATPase expression, suggesting that PEN is only a partial ligand of GPR83. By contrast, the renal specific siRNA-mediated silencing of pro-SAAS in mice increased SBP (130±4 vs control wild-type 100±1 mmHg, n=3/group; P&lt;0.05) that was associated with decreased urinary Na + excretion (P&lt;0.05) and increased renal expressions of NHE3, SGLT2, NaPi2, NKCC2, NCC, and Na + /K + -ATPase (P&lt;0.05 all). Renal expression of GRK4, another regulator of BP was also increased. Renal pro-SAAS expression was increased by a high salt diet (4%NaCl) in non-transgenic mice but was decreased in the salt-sensitive GRK4 486V mice on the same diet. Our results suggest that pro-SAAS peptides may be involved in the development of salt sensitivity through gene-gene interaction between GPR83 and GRK4 486.

Characterization of intracellular buffering power in human induced pluripotent stem cells and the loss of pluripotency is delayed by acidic stimulation and increase of NHE1 activity.

The homeostasis of intracellular pH (pHi ) affects many cellular functions. Our previous study has established a functional and molecular model of the active pHi regulators in human induced pluripotent stem cells (hiPSCs). The aims of the present study were to further quantify passive pHi buffering power (β) and to investigate the effects of extracellular pH and Na+ -H+ exchanger 1 (NHE1) activity on pluripotency in hiPSCs. pHi was detected by microspectrofluorimetry with pH-sensitive dye-BCECF. Western blot, immunofluorescence staining, and flow cytometry were used to detect protein expression and pluripotency. Our study in hiPSCs showed that (a) the value of total (βtot ), intrinsic (βi ), and CO2 -dependent ( ) buffering power all increased while pHi increased; (b) during the spontaneous differentiation for 4 days, the β values of βtot and changed in a tendency of decrease, despite the absence of statistical significance; (c) an acidic cultured environment retained pluripotency and further upregulated expression and activity of NHE1 during spontaneous differentiation; (d) inhibition on NHE1 activity promoted the loss of pluripotency. In conclusion, we, for the first time, established a quantitative model of passive β during differentiation and demonstrated that maintenance of NHE1 at a higher level was of critical importance for pluripotency retention in hiPSCs.

NHE1 Mediates 5-Fu Resistance in Gastric Cancer via STAT3 Signaling Pathway.

BackgroundSeveral recent studies have addressed the role of Na+/H+ exchanger isoform 1 (NHE1) in tumor cell growth and apoptosis, including in gastric cancer. However, the role of NHE1 expression related to the 5-Fu resistance in gastric cancer has not been investigated.MethodsThe expression of NHE1 was examined by qPCR in the SGC7901/5-FU cell line and its parental cell line. pcDNA3.1-NHE1 and NHE1-siRNA were transfected to SGC7901/5-FU resistance cells and cell apoptosis was detected via TUNEL assay. The upstream activators in NHE1 mediated 5-Fu resistant gastric cancer cells were detected by Western blot and immunofluorescent.ResultsA significant increase of the expression of NHE1 was observed in SGC7901 5-FU resistance cells compared to the GES-1 and SGC7901 cell line. NHE1 can suppress the cell apoptosis of SGC7901 5-FU resistance cells and involved in cell cycle. Also, the migration and invasion of SGC7901 5-FU resistance cells were promoted by NHE1. NHE1 also increases the intracellular pH. The results of Western blot analysis showed that NHE1 overexpression induced an increase in the expression of phosphorylated activator transcription factor 3 (pSTAT3). The more obvious phosphorylated level was shown in the phosphorylated STAT3 at pSTAT3tyr705. Further investigations revealed that the constitutive activation of STAT3 may be induced by JAK1 and JAK2, and thus effect the 5-FU resistance by regulating NHE1.DiscussionIn summary, our findings provided evidence that NHE1 contributed to 5-Fu resistance in gastric cancer cells by regulating the JAK/STAT3 pathway. Therefore, NHE1 can be a useful marker for predicting and monitoring 5-Fu resistance.