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 (ßi) 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.

Expression, Localization and Functional Activity of the Major Na⁺/H⁺ Exchange Isoforms Expressed in the Intestinal Cell Line Caco-2BBe.

BACKGROUND/AIMS: Enterocytes express a number of NHE isoforms with presumed localization in the apical (NHE2, 3 and 8) or basolateral (NHE1) membrane. Functional activity and localization of enterocyte NHE isoforms were assessed using fully differentiated Caco-2BBe cells, whose genetic expression profile closely resembles mature enterocytes. METHODS: The activity of the different NHEs was analyzed by fluorometric pHi-metry in a perfusion chamber with separate apical and basolateral perfusion, using specific inhibitors and shRNA knockdown of NHE2. The expression of the NHEs and of other relevant acid extrusion transporters was quantified by qPCR. RESULTS: Quantitative comparison of the mRNA expression levels of the different NHE isoforms in 14 day-differentiated Caco-2BBe cells showed the following order: NHE2>NHE8>NHE3>NHE1. Acid-activated NHE exchange rates in the basolateral membrane were >6-fold higher than in the apical membrane. 79 ± 3 % of the acid-activated basolateral Na⁺/H⁺ exchange rate displayed a NHE1-typical inhibitor profile, and no NHE2/3/8 typical activity could be observed. Analysis of the apical Na⁺/H⁺ exchange rates revealed that approximately 51 ± 3 % of the total apical activity displayed a NHE2/8-typical inhibitor profile and 31 ± 6 % a NHE3-typical inhibitor profile. Because no selective NHE2 inhibitor is available, a stable NHE2 knockdown cell line (C2NHE2KD) was generated. C2NHE2KD displayed a reduced NHE2-typical apical Na⁺/H⁺ exchange rate and maintained a lower steady-state pHi, despite high expression levels of other acid extruders, in particular NBCn1 (Slc4a7). CONCLUSION: Differentiated Caco-2BBe cells display particularly high mRNA expression levels of NHE2, which can be functionally identified in the apical membrane. Although at low intracellular pH, NHE2 transport rate was far lower than that of NHE1. NHE2 activity was nevertheless essential for the maintenance of the steady-state pHi of these cells.

Alleviative effect of fucoxanthin-containing extract from brown seaweed Laminaria japonica on renal tubular cell apoptosis through upregulating Na+/H+ exchanger NHE1 in chronic kidney disease mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Brown seaweed is a common food for Asians, and the bioactive ingredient fucoxanthin exerts anti-apoptotic activities in several cell types. Renal tubular cell apoptosis is one of the common cellular events leading to renal fibrosis and chronic kidney disease (CKD). However, the influence of fucoxanthin-containing brown seaweed extract on CKD is still unknown. We intended to evaluate the inhibitory effect of fucoxanthin-containing extract from brown seaweed on renal apoptosis under CKD condition and its molecular mechanism. MATERIALS AND METHODS: The fucoxanthin-containing brown seaweed extract (LJE) was prepared from Laminaria japonica. We investigated how LJE influences on both doxorubicin-treated rat renal tubular cells (NRK-52E) and the renal symptoms of nephrectomy-induced CKD mice. RESULTS: LJE inhibited doxorubicin-induced apoptosis and upregulated Na+/H+ exchanger isoform 1 (NHE1) expression in NRK-52E cells, which were blocked by the NHE1 inhibitor cariporide. LJE also upregulated peroxisome proliferator-activated receptor alpha (PPARα). PPARα siRNA transfection inhibited LJE-induced NHE1 expression and anti-apoptotic effect. In CKD mice, LJE increased NHE1 expression in renal tubules and reduced apoptotic renal tubular cells, but not in PPARα knockout mice. The inhibitory effect of LJE on apoptosis also reduced renal tubulointerstitial fibrosis and improved renal function in CKD mice. CONCLUSION: We demonstrated that LJE inhibits renal apoptosis via NHE1 upregulation. The anti-apoptotic effect of LJE also improves renal function in CKD mice. Therefore, fucoxanthin-containing brown seaweed may have a therapeutic potential for CKD patients.

Amelioration of Lipopolysaccharide-Induced Acute Lung Injury in Rats by Na-H Exchanger-1 Inhibitor Amiloride Is Associated with Reversal of ERK Mitogen-Activated Protein Kinase.

BACKGROUND: Na-H exchanger-1 (NHE-1) is expressed in the lung of rats. Accumulating evidence shows that Na-H exchangers are involved in inflammation. Amiloride, an inhibitor of NHE-1, inhibits the activation of macrophages and endothelial cells and reduces their production of cytokines. Since these processes have been implicated in acute lung injury (ALI) induced by lipopolysaccharide (LPS), we examined the protective effect of amiloride on ALI induced by LPS in rats. MATERIAL AND METHODS: ALI in specific pathogen-free male Sprague-Dawley rats was induced by an intravenous injection of 6 mg/kg LPS. Amiloride pretreated rats received an intravenous injection of 10 mg/kg amiloride 30 min before the administration of LPS. Controls received normal saline in a similar manner. All animals were sacrificed 6 h after LPS or normal saline administration. The degree of ALI was assessed by wet-to-dry weight ratio (W/D) and lung histological examination. Neutrophilic infiltration was determined by myeloperoxidase (MPO) activity in lung tissue. Concentrations of total protein (TP), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein-2 (MIP-2) in bronchoalveolar lavage fluid (BALF) were also measured. Expression of NHE-1 and mitogen-activated protein kinase (MAPK) p38, p-p38, ERK, and p-ERK was evaluated by western blot analysis. RESULTS: Pretreatment with amiloride significantly reduced the increase in W/D, ALI score, lung tissue MPO activity, concentrations of TP, TNF-α, and MIP-2 in BALF, resulting in attenuation of ALI induced by LPS. Meanwhile, levels of NHE-1 and p-ERK proteins were reversed, whereas that of p-p38 was not. CONCLUSIONS: These findings suggest that NHE-1 inhibitor amiloride could attenuate ALI induced by LPS in rats. This effect is mediated through reversal of ERK.