Increased NHE Activity Research Articles

Zinc-alpha2-glycoprotein modulates blood pressure by regulating renal lipid metabolism reprogramming - mediated urinary Na+ excretion in hypertension.

Organs modulating blood pressure are associated with a common cytokine known as adipokines. We chose Zinc-alpha2-glycoprotein (ZAG) due to its prioritized transcriptional level in the database. Previous studies showed that ZAG is involved in metabolic disorders. The aim of this study was to investigate its role in hypertension. Serum ZAG levels were assessed in hypertensive and healthy participants. Blood pressure was monitored in Azgp1-/- mice and other animal models by 24-hour ambulatory implanted telemetric transmitters and tail-cuff method. Multi-omics analysis of proteomics and metabolomics were performed to explore possible mechanisms. Serum ZAG levels were significantly decreased and associated with morning urine Na+ excretion in hypertensive participants in a cross-sectional study. This study firstly reported that Azgp1-/- mice exhibited increased blood pressure and impaired urinary Na+ excretion, which were restored by AAV9-mediated renal tubule Azgp1 rescue. Azgp1 knockout caused the reprogramming of renal lipid metabolism, and increased Na+/H+-exchanger (NHE) activity in the renal cortex. Administration with a NHE inhibitor EIPA reversed the impaired urinary Na+ excretion in Azgp1-/- mice. Moreover, the activity of carnitine palmitoyltransferase 1 (CPT1), a key enzyme of fatty acid β-oxidation, was decreased, and the levels of malonyl-CoA, an inhibitor of CPT1, were increased in renal cortex of Azgp1-/- mice. Renal Cpt1 rescue improved urinary Na+ excretion and blood pressure in Azgp1-/- mice, accompanied by decreased renal fatty acid levels and NHE activity. Finally, administration of recombinant ZAG protein improved blood pressure and urinary Na+ excretion in SHRs. Deficiency of Azgp1 increased the malonyl CoA-mediated inhibition of CPT1 activity, leading to renal lipid metabolism reprogramming, resulting in accumulated fatty acids and increased NHE activity, subsequently decreasing urinary Na+ excretion and causing hypertension. These findings may provide a potential kidney-targeted therapy in the prevention and treatment of hypertension.

Abstract We120: NHE1 Regulates Apoptosis Susceptibility in Sugen-Hypoxia Pulmonary Arterial Smooth Muscle Cells via Modulation of CHOP

Pulmonary hypertension (PH) is caused by lung vascular remodeling involving increased pulmonary arterial smooth muscle cell (PASMC) proliferation and apoptosis resistance. Na +/ H + exchanger (NHE) isoform 1 (NHE1) is a membrane protein regulating intracellular pH (pH i ) via movement of H + . Our lab found increased NHE activity is associated with hyperproliferation in PASMCs from the Sugen Hypoxia (SuHx) animal model of PH. In this study, we tested the hypothesis that increased NHE1 activity also causes apoptosis resistance in SuHx PASMCs via dysregulation of pH i . PASMCs isolated from distal pulmonary arteries of control and SuHx rats were treated with NHE inhibitor, [5-(N-ethyl-N-isopropyl)-amiloride; EIPA;1 uM], NHE1 inhibitor [cariporide;10 uM] or vehicle for 24 h before analyzing cell apoptosis via Hoechst staining under baseline and stimulated (250 uM H 2 O 2 ) conditions. Control PASMCs were infected with adenovirus containing green fluorescent protein (GFP), wild-type NHE1 (WT) or NHE1 lacking ion translocation activity (E262I). Protein expression and localization were assessed via immunoblotting. NHE activity was assessed from Na + -dependent recovery following an ammonium pulse. At baseline, apoptosis in SuHx PASMCs was similar to control cells but following H 2 O 2 stimulation , SuHx PASMCs were resistant to apoptosis (p<0.001). EIPA increased basal and stimulated apoptosis in SuHx cells. Surprisingly, treatment with cariporide did not alter SuHx apoptosis, suggesting that EIPA but not cariporide was able to sensitize SuHx cells to apoptosis, despite both drugs significantly inhibiting NHE activity, suggesting EIPA affects PASMC apoptosis through a mechanism other than pH i regulation. To test this possibility, control PASMCs were infected with GFP, WT or E262I. NHE activity was increased in cells with WT, but not E262I, when compared to GFP (p<0.001). Overexpression of either WT or E262I inhibited apoptosis following treatment with H2O2 (p<0.001). We next explored CAAT/enhancer binding protein homologous transcription factor (CHOP), known to be involved in induction of apoptosis. CHOP expression was increased by H2O2 in control cells but was decreased in SuHx PASMCs. EIPA increased CHOP levels in SuHx cells, whereas, expression of both WT and E262I constructs inhibited the H 2 O 2 -induced CHOP increase in control PASMCs. Our results indicate that NHE1 regulates apoptosis susceptibility in PASMCs during PH via modulation of CHOP rather than through pH i regulation.

Na+/H+ exchanger isoform 1 (NHE1) confers resistance to apoptosis in pulmonary arterial smooth muscle cells (PASMCs)

Pulmonary hypertension (PH) is a condition caused by remodeling of the lung vasculature due to abnormal cell turnover involving increased cell proliferation and resistance to apoptosis. The mechanisms controlling abnormal cell growth and survival are incompletely understood. We previously found that Na+/H+ exchanger (NHE) activity is increased in PASMCs from PH patients and the Sugen Hypoxia (SuHx) animal model of PH. NHE isoform 1 (NHE1) is a membrane protein that regulates intracellular pH (pHi) and cell volume via export of H+ and import of Na+. This protein also has a cytosolic tail region shown to be involved in non-canonical functions such as cytoskeletal interactions and cell signaling via protein-protein interactions. NHE1 has been associated with increased PASMC proliferation; however, it is unclear if increased NHE activity, dysregulation of pHi or cytoskeletal interactions are involved in resistance to apoptosis. In this study, we tested the hypothesis that increased expression of NHE1 leads to apoptosis resistance in PASMCs via dysregulation of pHi. We first used the rat SuHx model of PH in which male Wistar rats receive a single subcutaneous injection of the vascular endothelial growth factor receptor inhibitor, SU5416, followed by 3 wks of exposure to hypoxia (10% O2) and 2 wks of normoxia. PASMCs were isolated from distal pulmonary arteries of control and SuHx Wistar rats and treated with the NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 1 uM), or vehicle for 24 h before analyzing cell apoptosis via Hoechst staining under baseline and stimulated conditions (250 uM H2O2). Endoplasmic reticulum stress was measured using the fluorescent stain, Thioflavin T (ThT). In some experiments, cells from control rats were infected with adenovirus containing green fluorescent protein (AdGFP; control for infection), wild-type NHE1 (AdNHE1-WT) or NHE1 mutants: AdNHE1-E262I lacking ion translocation activity and AdNHE1-EM lacking the ezrin binding site which prevents cytoskeletal interactions. NHE1 protein expression and localization were assessed via immunoblotting (total and surface proteins). Effcacy of Na+/H+ translocation was assessed from Na+-dependent recovery following an ammonium pulse. At baseline, despite increased ThT levels in SuHx cells, apoptosis was not different compared to control cells, indicating SuHx cells have suppressed apoptotic response to increased ER stress. Following treatment with EIPA, apoptosis was increased in SuHx cells, possibly indicating they became sensitized to the high levels of ER stress and were able to proceed into the apoptotic pathway. Similarly, following treatment with H2O2, SuHx PASMCs showed resistance to apoptosis compared to control cells, which was alleviated with pre-treatment with EIPA. In control cells, wild-type NHE1 and NHE1 mutants were expressed in similar amounts which led to an expected increase in NHE activity for WT and NHE1-EM, but not for NHE1-E262I when compared to GFP. We found that enhanced expression of NHE1-WT was suffcient to inhibit apoptosis following treatment with H2O2. Interestingly, both NHE1 mutants also significantly decreased PASMC apoptosis following H2O2 stimulation. Our results indicate NHE1 is necessary for apoptosis resistance in PASMCs from animals with PH and that increased expression is suffcient to confer resistance to apoptosis in control PASMCs. Additionally, these results suggest neither pHi regulation nor interaction between NHE1 and ezrin are essential for NHE1-induced apoptosis resistance, indicating another mechanism through which NHE1 confers resistance to apoptosis in PH. Based on these findings, we speculate that development of NHE1-related therapeutic targets could potentially restore cell apoptosis and reverse disease. AHA 23PRE1022720, HL159906, HL073859. 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.

Rimeporide, a first in class Na/H exchanger-1 inhibitor, ameliorates right ventricular dysfunction and pulmonary arterial hypertension in a SuHx model

Abstract Background Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and vascular remodeling leading to right ventricular (RV) failure. Although RV function is the major prognostic factor in PAH, no RV-specific therapies exist. The Na+/H+ exchanger type 1 (NHE1) regulates the intra- and extracellular pH balance mainly through the 1:1 exchange of intracellular H+ with extracellular Na+. Increased NHE-1 activity is involved in several diseases, including hypoxic PAH. Inhibition of NHE-1 activity attenuates pulmonary vascular remodeling in rodents exposed to hypoxia but its potential direct contribution in RV remodeling remains unclear. Aim In the current study, we aimed to investigate the effect of NHE-1 inhibition by Rimeporide a preclinical hypoxic models of PH: Sugen5416/hypoxia. Methods Sugen/hypoxia model: PAH was induced in adult Sprague-Dawley rats with a single injection of SU5416 (20 mg/kg) followed by 3 weeks of hypoxia (10% O2; Sugen/Hypoxia model: SuHx) and then return to normoxia. Control rats were injected with equal volume of vehicle. At week 5 post-injection, control and SuHx rats were treated with rimeporide (100 mg/Kg daily in drinking water, n=10/group) or placebo (n=11/group) for 3 weeks. Echocardiography and blood sampling were performed. RV phenotype was monitored by echocardiography and hemodynamic analysis. Lung and heart were collected for histological and protein expression analysis, such as NHE-1 (target of Rimeporide). Results SuHx caused severe PAH and RV dysfunction. RV dysfunction was accompanied by an increase in RV hypertrophy and RV fibrosis and PA remodeling (increase in the arteriolar wall thickness and fibrosis). A marked increase in macrophages was found in lung and RV tissues but not in LV. Moreover, Western blot analysis showed that RV protein levels of NHE-1 was higher in SuHx rats than Normoxia rats. Treatment with Rimeporide reversed SuHx-induced PAH as well as RV dysfunction and blunted pulmonary and RV inflammation, RV NHE-1 protein expression. Conclusion Our results indicate that NHE-1 levels are increased in RV tissues from SuHx-induced PAH rats. Rimeporide, through NHE-1 inhibition, might be a potential new target for treatment of RV dysfunction and PAH.

Luseogliflozin inhibits high glucose-induced TGF-β2 expression in mouse cardiomyocytes by suppressing NHE-1 activity.

ObjectiveSodium-glucose cotransporter-2 (SGLT2) inhibitors exhibit cardioprotective properties in patients with diabetes. However, SGLT2 is not expressed in the heart, and the underlying molecular mechanisms are not fully understood. We investigated whether the SGLT2 inhibitor luseogliflozin exerts beneficial effects on high glucose-exposed cardiomyocytes via the suppression of sodium-hydrogen exchanger-1 (NHE-1) activity.MethodsMouse cardiomyocytes were incubated under normal or high glucose conditions with vehicle, luseogliflozin, or the NHE-1 inhibitor cariporide. NHE-1 activity and gene expression were evaluated by the SNARF assay and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. Six-week-old male db/db mice were treated with vehicle or luseogliflozin for 6 weeks, and the hearts were collected for histological, RT-PCR, and western blot analyses.ResultsHigh glucose increased NHE-1 activity and transforming growth factor (Tgf)-β2 mRNA levels in cardiomyocytes, both of which were inhibited by luseogliflozin or cariporide, whereas their combination showed no additive suppression of Tgf-β2 mRNA levels. Luseogliflozin attenuated cardiac hypertrophy and fibrosis in db/db mice in association with decreased mRNA and protein levels of TGF-β2.ConclusionsLuseogliflozin may suppress cardiac hypertrophy in diabetes by reducing Tgf-β2 expression in cardiomyocytes via the suppression of NHE-1 activity.

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.

Effects of the Na+/H+ Ion Exchanger on Susceptibility to COVID-19 and the Course of the Disease.

The Na+/H+ ion exchanger (NHE) pumps Na+ inward the cell and H+ ion outside the cell. NHE activity increases in response to a decrease in intracellular pH, and it maintains intracellular pH in a narrow range. Patients with obesity, diabetes, and hypertension and the elderly are prone to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The angiotensin II (Ang II) level is high in chronic diseases such as diabetes, hypertension, and obesity. Ang II is the main stimulator of NHE, and an increased Ang II level causes prolonged NHE activation in these patients. The long-term increase in NHE activity causes H+ ions to leave the cell in patients with diabetes, hypertension, and obesity. Increasing H+ ions outside the cell lead to an increase in oxidative stress and reactive oxygen species. H+ ion flows into the cell due to the increased oxidative stress. This vicious circle causes intracellular pH to drop. Although NHE is activated when intracellular pH decreases, there is prolonged NHE activation in chronic diseases such as aforementioned. Novel coronavirus disease 2019 (COVID-19) progression may be more severe and mortal in these patients. SARS-CoV-2 readily invades the cell at low intracellular pH and causes infection. The renin-angiotensin system and NHE play a vital role in regulating intracellular pH. The reduction of NHE activity or its prolonged activation may cause susceptibility to SARS-CoV-2 infection by lowering intracellular pH in patients with diabetes, hypertension, and obesity. Prolonged NHE activation in these patients with COVID-19 may worsen the course of the disease. Scientists continue to investigate the mechanism of the disease and the factors that affect its clinical progression.

Role of Na+/H+ Exchanger 1 in Regulating Resistance to Apoptosis in Pulmonary Arterial Smooth Muscle Cells

Vascular remodeling within the pulmonary arterioles is a key component of pulmonary arterial hypertension (PAH). In a rat model of severe PAH, the SU5416‐Hypoxia (SuHx) model, pulmonary arterial smooth muscle cells (PASMCs) demonstrate resistance to apoptosis and contribute to vascular remodeling. Na+/H+ exchange (NHE) is increased in SuHx PASMCs; however, few studies have elucidated the functional role of NHE in these cells. Previous studies have shown a link between NHE and the expression of CHOP, a critical mediator of apoptosis. Therefore, we tested the hypothesis that altering NHE1 expression and or activity is sufficient to modulate apoptosis in PASMCs. PASMCs were isolated from distal pulmonary arteries dissected from vehicle‐treated, male, Wistar rats or rats exposed to hypoxia after an injection of SU5416 (SuHx rats). PASMCs were treated with vehicle, a combination of a pharmacologic inhibitor of NHE (EIPA) and hydrogen peroxide (H2O2), an apoptosis‐inducing agent, or either EIPA or H2O2 alone. Apoptosis was measured via Hoechst staining. In other experiments, control PASMCs were infected with a viral construct to overexpress wild‐type NHE1 (AdNHE1), a translocation‐disabled NHE1 mutant (AdE266I), or green fluorescent protein (AdGFP) as a control. NHE activity was measured via the ammonium‐pulse technique. SuHx PASMCs exhibited decreased CHOP expression and minimal apoptosis rates in response to H2O2 when compared to control PASMCs. EIPA had no effect on unstimulated apoptosis in control cells, but induced apoptosis in SuHx PASMCs at baseline and sensitized the cells to H2O2. AdNHE1, but not AdE266I, increased NHE1 activity. Interestingly, both AdNHE1 and AdE266I conferred a resistance to H2O2‐induced apoptosis when compared to the AdGFP‐infected cells. Overall, these findings suggest that increased NHE1 may be responsible for attenuating apoptotic responses in SuHx PASMCs and that increasing NHE1 levels in normal PASMCs is sufficient to promote increased survival via an ion‐translocation‐independent function.Support or Funding InformationSupported by NIH grants HL073859, HL126514 and HL084762

Aquaporin-2 and Na+ /H+ exchanger isoform 1 modulate the efficiency of renal cell migration.

Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin β1 trafficking and the turnover of focal adhesions. The aim of this study was to investigate whether AQP2 also works in cooperation with Na+ /H+ exchanger isoform 1 (NHE1), another well-known protein involved in the regulation of cell migration. Our results showed that the lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and areas of focal adhesions and more alkaline intracellular pH due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2, or its physically-associated calcium channel TRPV4, significantly reduced lamellipodia NHE1 activity. NHE1 blockage significantly reduced the rate of cell migration, the number of lamellipodia, and the assembly of F-actin only in AQP2-expressing cells. Our data suggest that AQP2 modulates the activity of NHE1 through its calcium channel partner TRPV4, thereby determining pH-dependent actin polymerization, providing mechanical stability to delineate lamellipodia structure and defining the efficiency of cell migration.

Rho kinase and Na+ /H+ exchanger mediate endothelin-1-induced pulmonary arterial smooth musclecell proliferation and migration.

Excessive production of endothelin‐1 (ET‐1) has been observed in almost all forms of pulmonary hypertension. ET‐1, a highly potent vasoconstrictor, can also potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. Increasing evidence indicates that alkalinization of intracellular pH (pH i), typically due to activation of Na+/H+ exchange (NHE), is associated with enhanced PASMC proliferation and migration. We recently demonstrated that application of exogenous ET‐1 increased NHE activity in murine PASMCs via a mechanism requiring Rho kinase (ROCK). However, whether ROCK and/or increased NHE activity mediate ET‐1‐induced migration and proliferation in PASMCs remains unknown. In this study, we used fluorescent microscopy in transiently cultured PASMCs from distal pulmonary arteries of the rat and the pH‐sensitive dye, BCECF‐AM, to measure changes in resting pH i and NHE activity induced by exposure to exogenous ET‐1 (10−8 mol/L) for 24 h. Cell migration and proliferation in response to ET‐1 were also measured using Transwell assays and BrdU incorporation, respectively. We found that application of exogenous ET‐1 had no effect on NHE1 expression, but increased pH i, NHE activity, migration, and proliferation in rat PASMCs. Pharmacologic inhibition of NHE or ROCK prevented the ET‐1‐induced changes in cell function (proliferation and migration). Our results indicate that ET‐1 modulates PASMC migration and proliferation via changes in pH i homeostasis through a pathway involving ROCK.

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

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

The Na+/H+ exchanger contributes to increased smooth muscle proliferation and migration in a rat model of pulmonary arterial hypertension.

Increased muscularity of small pulmonary vessels, involving enhanced proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a key component of the vascular remodeling underlying the development of pulmonary hypertension (PH). Stimuli such as growth factors and hypoxia induce PASMC alkalinization, proliferation, and migration through upregulation of the Na+/H+ exchanger (NHE), inhibition of which prevents the development of hypoxia‐induced vascular remodeling and PH. We wanted to explore whether NHE was also necessary for pathologic PASMC proliferation and migration in a model of pulmonary arterial hypertension (PAH), a severe form of PH not associated with persistent hypoxia. PASMCs were isolated from rats exposed to SU5416‐hypoxia (SuHx) followed by return to normoxia and from vehicle controls. We measured resting intracellular pH (pH i) and NHE activity using the pH‐sensitive fluorescent dye BCECF‐AM. PASMC proliferation and migration were assessed using BrdU incorporation and transwell filters, respectively. NHE activity was increased in SuHx PASMCs, although resting pH i was unchanged. SuHx PASMCs also exhibited increased proliferation and migration relative to controls, which was attenuated in the setting of pharmacologic inhibition of NHE. Our findings suggest that increased NHE activity contributes to pathologic PASMC function in the SuHx model of PAH, although this effect does not appear to be mediated by global changes in pH i homeostasis.

Quercetin-modulated erythrocyte membrane sodium-hydrogen exchanger during human aging: correlation with ATPase’s

Content: Quercetin uptake by erythrocytes is rapid. The sodium-hydrogen exchanger (NHE) is a secondary active transporter, regulating intracellular pH, Na+ concentration and cell volume. Objective: The aim of present study was to investigate NHE as a function of human age and effect of quercetin on its activity. The NHE activity was correlated with erythrocytes ATPases. Materials and methods: We analyzed normal, healthy subjects of both sexes (20–82 years). NHE activity was estimated in terms of amiloride-sensitive H+-efflux from acid-loaded cells. Results: A significant age-dependent increase in NHE activity was observed during aging in humans. Concentration (10−3 M to 10−8 M)-dependent in vitro treatment with quercetin causes inhibition of NHE activity. The Na+/K+ -ATPase (r = 0.8882) and Ca2+-ATPase (r = 0.9540) activities positively correlated with it. Discussion and conclusion: The present data show an additional mechanism where dietary flavonoids may exerts beneficial effect during aging.

Abstract 087: Human Stomach Cell Gastrin Inhibits Renal NHE3 and NaKATPase in Concert With the Renal D1R

The digestive track secretes gastrin in response to sodium ingestion stimulating increased renal sodium excretion. We previously showed that gastrin secreted by human colon cancer cells (SW626) can bind to cell surface cholecystokinin receptors on renal proximal tubule cells (RPTC), modulating the natriuretic dopaminergic system. We tested the hypothesis that a similar gastro/renal axis exists in humans. Novel human stomach antrum G-cell lines from 6 separate individuals were isolated and each shown to express gastrin mRNA and protein, and bind Phaseolus vulgaris Leucoagglutinin (PHA-l) by fluorescence lectin affinity (marker for gastrin secreting cells). It was determined that the D1 receptor (D1R) is also found on G-Cells. In human stomach tissue, gastrin expression was increased by Fenofibrate treatment, (PPARα agonist, p= 0.07, in 3 live ex-vivo cultured human stomachs). Moreover, we tested the effect of gastrin on CCKB2 receptors in human RPTC. Gastrin (100 nM 15 min.) increased RPTC phospholipace-C (PLC) activity by 1.07± 0.01 fold, N=35, p<0.0001, using a PLC-FRET biosensor, CYPHR, but did not increase cAMP levels using the specific cAMP-FRET biosensor, ICUE-YR. Both gastrin and SKF83822 (100 nM, a cAMP specific D1R agonist, 15 min) alone reduced sodium influx into RPTC via NHE3 (from VEH 100± 4.7% to 73.5± 6.2% or 74.1± 4.3% respectively N=6, p<0.05), but gastrin along with SKF83822 decreased sodium influx more than either alone (57.2 ± 5.8% N=6, p<0.05). Sodium efflux via NaKATPase was reduced by SKF83822 (from VEH 100± 3.9% to 84.2± 3.3%, N=6, p<0.05), but not gastrin alone, however SKF83822 along with gastrin reduced sodium efflux more than SKF83822 alone (72.3±5.1% vs N=6, p<0.05). Additionally we found that Angiotensin II (AngII, 10 nM, 15 min.) increased NHE3 activity (12.3± 3.6% N=6, p<0.01) and this increase was completely blocked by gastrin (N=6, p<0.01). The PLC inhibitor U73122 reversed the inhibitory effect of gastrin on NHE3 and NaKATPase. Gastrin was also found to decrease the amount of fluorescent AngII binding to RPTCs (27.4± 6.1% N=6, p<0.01) and this decrease was completely blocked by U73122. Thus, both stomach gastrin and the renal D1R inhibit NHE3 and NaKATPase, to increase sodium elimination from the body after salt is ingested.

Short- and long-term regulation of intestinal Na+/H+ exchange by Toll-like receptors TLR4 and TLR5.

Inappropriate activation of pattern recognition receptors has been described as a potential trigger in the development of inflammatory bowel disease (IBD). In this study, we evaluated the activity and expression of Na(+)/H(+) exchanger (NHE) subtypes in T84 intestinal epithelial cells during Toll-like receptor 4 (TLR4) activation by monophosphoryl lipid A and TLR5 by flagellin. NHE activity and intracellular pH were evaluated by spectrofluorescence. Additionally, kinase activities were evaluated by ELISA, and siRNA was used to specifically inhibit adenylyl cyclase (AC). Monophosphoryl lipid A (MPLA) (0.01-50.00 μg/ml) and flagellin (10-500 ng/ml) inhibited NHE1 activity in a concentration-dependent manner (MPLA short term -25.2 ± 5.0%, long term -31.9 ± 4.0%; flagellin short term -14.9 ± 2.0%, long term -19.1 ± 2.0%). Both ligands triggered AC3, PKA, PLC, and PKC signal molecules. Long-term exposure to flagellin and MPLA induced opposite changes on NHE3 activity; flagellin increased NHE3 activity (∼10%) with overexpression of membrane protein, whereas MPLA decreased NHE3 activity (-17.3 ± 3.0%). MPLA and flagellin simultaneously had synergistic effects on NHE activity. MPLA and flagellin impaired pHi recovery after intracellular acidification. The simultaneous exposure to MPLA and flagellin induced a substantial pHi reduction (-0.55 ± 0.03 pH units). Activation of TLR4 and TLR5 exerts marked inhibition of NHE1 activity in intestinal epithelial cells. Transduction mechanisms set into motion during TLR4-mediated and long-term TLR5-mediated inhibition of NHE1 activity involve AC3, PKA, PLC, and PKC. However, short- and long-term TLR4 activation and TLR5 activation might use different signaling pathways. The physiological alterations on intestinal epithelial cells described here may be useful in the development of better IBD therapeutics.

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss.

Diarrhea is one of the troublesome complications of diabetes, and the underlying causes of this problem are complex. Here, we investigated whether altered electrolyte transport contributes to diabetic diarrhea. We found that the expression of Na+/H+ exchanger NHE3 and several scaffold proteins, including NHE3 regulatory factors (NHERFs), inositol trisphosphate (IP₃) receptor-binding protein released with IP₃ (IRBIT), and ezrin, was decreased in the intestinal brush border membrane (BBM) of mice with streptozotocin-induced diabetes. Treatment of diabetic mice with insulin restored intestinal NHE3 activity and fluid absorption. Molecular analysis revealed that NHE3, NHERF1, IRBIT, and ezrin form macrocomplexes, which are perturbed under diabetic conditions, and insulin administration reconstituted these macrocomplexes and restored NHE3 expression in the BBM. Silencing of NHERF1 or IRBIT prevented NHE3 trafficking to the BBM and insulin-dependent NHE3 activation. IRBIT facilitated the interaction of NHE3 with NHERF1 via protein kinase D2-dependent phosphorylation. Insulin stimulated ezrin phosphorylation, which enhanced the interaction of ezrin with NHERF1, IRBIT, and NHE3. Additionally, oral administration of lysophosphatidic acid (LPA) increased NHE3 activity and fluid absorption in diabetic mice via an insulin-independent pathway. Together, these findings indicate the importance of NHE3 in diabetic diarrhea and suggest LPA administration as a potential therapeutic strategy for management of diabetic diarrhea.

Renal nerve stimulation leads to the activation of the Na+/H+exchanger isoform 3 via angiotensin II type I receptor

Renal nerve stimulation at a low frequency (below 2 Hz) causes water and sodium reabsorption via α1-adrenoreceptor tubular activation, a process independent of changes in systemic blood pressure, renal blood flow, or glomerular filtration rate. However, the underlying mechanism of the reabsorption of sodium is not fully understood. Since the sympathetic nervous system and intrarenal ANG II appear to act synergistically to mediate the process of sodium reabsorption, we hypothesized that low-frequency acute electrical stimulation of the renal nerve (ESRN) activates NHE3-mediated sodium reabsorption via ANG II AT1 receptor activation in Wistar rats. We found that ESRN significantly increased urinary angiotensinogen excretion and renal cortical ANG II content, but not the circulating angiotensinogen levels, and also decreased urinary flow and pH and sodium excretion via mechanisms independent of alterations in creatinine clearance. Urinary cAMP excretion was reduced, as was renal cortical PKA activity. ESRN significantly increased NHE3 activity and abundance in the apical microvillar domain of the proximal tubule, decreased the ratio of phosphorylated NHE3 at serine 552/total NHE3, but did not alter total cortical NHE3 abundance. All responses mediated by ESRN were completely abolished by a losartan-mediated AT1 receptor blockade. Taken together, our results demonstrate that higher NHE3-mediated proximal tubular sodium reabsorption induced by ESRN occurs via intrarenal renin angiotensin system activation and triggering of the AT1 receptor/inhibitory G-protein signaling pathway, which leads to inhibition of cAMP formation and reduction of PKA activity.

P2X receptors trigger intracellular alkalization in isolated perfused mouse medullary thick ascending limb.

AimsExtracellular ATP is an important regulator of renal tubular transport. Recently, we found that basolateral ATP markedly inhibits Na+ and Cl− absorption in mouse medullary thick ascending limb (mTAL) via a P2X receptor. The underlying mechanism that mediates this ATP-dependent transport inhibition in mTAL is, however, unclear. The renal outer medullary K+ channel (ROMK) is sensitive to intracellular pH where a reduction leads to closing of ROMK. We speculated that P2X receptor stimulation in the TAL could lead to changes in pHi, leading to a reduction in NaCl transport.MethodsTo test this hypothesis, we measured pHi in single perfused mouse mTALs using the fluorescent ratiometric dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester.ResultsInterestingly, basolateral ATP (100 μm) caused a prominent, reversible intracellular alkalization of mTAL, with an average pHi increase of 0.14 ± 0.02 (n = 14). This was completely abolished by the P2X receptor antagonist periodate-oxidized ATP (50 μm). The P2X receptor-mediated intracellular alkalization required the activity of the apical Na+/H+ exchanger (NHE3). Typically, Gq-coupled receptors cause a significant acidification of tubular epithelial cells, which was confirmed in this study, by P2Y2 and Ca2+ sensing receptor stimulation.ConclusionThis study reports that stimulation of basolateral P2X receptors causes a substantial intracellular alkalization in the isolated perfused mouse mTAL. This intracellular alkalization is mediated through an increased apical NHE3 activity, similar to what we previously observed when tubular transport is inhibited with furosemide. This increased NHE3 activity causes H+ secretion in the mTAL and provides further support that the TAL is a site of urinary acidification.

Renal NHE expression and activity in neonatal NHE3- and NHE8-null mice.

Na(+)/H(+) exchanger (NHE)3 is the predominant NHE on the brush-border membrane of the proximal tubule in adult animals. NHE8 has been localized to the brush-border membrane of proximal tubules and is more highly expressed in neonates than in adult animals. However, the relative role of NHE8 in neonatal renal acidification is unclear. The present study examined if there was a compensatory increase in NHE3 in NHE8-null neonatal mice and whether there was a compensatory increase in NHE8 in NHE3-null neonatal mice. In addition, we examined whether wild-type, NHE3-null, and NHE8-null mice had an increase in NHE activity in response to metabolic acidosis. We found that at baseline, there was comparable renal NHE3 mRNA, total protein, and brush-border membrane protein abundance as in neonatal control and NHE8-null mice. There was comparable renal NHE8 mRNA, total protein, and brush-border membrane protein abundance in NHE3-null neonatal and control mice. Both NHE3- and NHE8-null mice had a comparable but lower rate of NHE activity than control mice. We next imposed metabolic acidosis in wild-type, NHE3-null, and NHE8-null mice. Acidemic NHE8-null mice had an increase in brush-border membrane vesicle NHE3 protein abundance and NHE activity compared with vehicle-treated mice. Likewise, NHE3-null mice had an increase in NHE8 brush-border membrane protein abundance and NHE activity in response to metabolic acidosis. In conclusion, both NHE3 and NHE8 likely play a role in neonatal acidification.

The adrenergic regulation of proximal tubular Na+/H+ exchanger 3 in the rat

This study in the anaesthetized rat investigated how renal sympathetic nerve activity and catecholamine release influenced NHE3 abundance and activity in proximal tubular brush border membranes using both in vivo and in vitro approaches. Renal excretory function and brush border NHE3 abundance and activity were measured in rat kidneys which underwent renal denervation, renal nerve electrical stimulation and renal infusion of phenylephrine and the NHE3 inhibitor S1661. NHE3 activity and cell surface abundance were also measured in primary cultures of proximal tubular cells treated with noradrenaline and prazosin. Acute renal denervation caused a natriuresis and diuresis, which occurred with a reduction in NHE3 abundance and activity in the brush border membranes. By contrast, low-level electrical stimulation of the renal innervation causing an antinatriuresis and antidiuresis increased NHE3 activity in the brush border membranes. Intrarenal infusion of phenylephrine caused an antinatriuresis and antidiuresis, while blockade of NHE3 activity, using local infusion of the blocker S1661, caused a natriuresis and diuresis. Exposure of primary cultures of proximal tubular cells to noradrenaline increased brush border NHE3 abundance and activity which was blocked by prior exposure to prazosin, indicating it as an α1 -adrenoceptor-mediated mechanism. Together, these findings demonstrate that the renal sympathetic nerves not only have a direct action to modulate tubular sodium reabsorption via stimulation of the NHE transporter, but also have an indirect effect, whereby NHE3 abundance is increased within the brush border membrane, thereby increasing the capacity for fluid reabsorption.