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Original article
Role of sodium-hydrogen exchanger isoform 1 in regulating hepatocyte apoptosis induced by hyperammonaemia
Papel de la isoforma 1 del intercambiador iónico de sodio/hidrógeno en la regulación de la apoptosis de hepatocitos inducida por hiperamoniaquemia
Peng Wanga,1, Xin Wanga,1,
Corresponding author
wang_x@aliyun.com

Corresponding author.
, Ling Lib, Quancheng Kanc, Zujiang Yuc, Rongfang Fenga, ZiXiao Chena, Yan Shia, Jinling Gaoa
a Department of Basic Medicine, Nursing College, Zhengzhou University, Zhengzhou, Henan, PR China
b Department of Palliative and Hospice Care, The Ninth People's Hospital of Zhengzhou, Henan, PR China
c Department of Infectious Disease, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, PR China
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Hepatic failure &#40;HF&#41; is a life threatening disease and is caused by a variety of factors&#44; which induce liver cell damage and liver dysfunction&#46; The mortality rate of HF is very high<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">1</span></a> and the complications associated with HF include hepatic encephalopathy&#44; hepatorenal syndrome and hemorrhaging&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">2</span></a> It has been proved that hyperammonaemia was involved in the pathogenesis of hepatic encephalopathy and it is commonly studied as the mechanism of encephalopathy&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">3</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">Recent studies&#44; including the results obtained from our laboratory&#44; have demonstrated that hyperammonemia has a direct adverse effect on hepatocytes and it is therefore both a cause and an effect of hepatic failure&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">4&#8211;6</span></a> But the effect of high blood ammonia on liver cell injury and its underlying mechanism remain unclear&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Proper cell function depends on the maintenance of intracellular and extracellular physicochemical parameters within certain physiological limits&#46; Such an important parameter is pH&#46;<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">7</span></a> In fact&#44; the activity of intracellular enzymes&#44; the interaction of cytoskeletal elements&#44; the rate at which cells grow and differentiate all depend on pHi&#46;<a class="elsevierStyleCrossRefs" href="#bib0200"><span class="elsevierStyleSup">8&#44;9</span></a> Cells can minimize significant pHi fluctuations through several H<span class="elsevierStyleSup">&#43;</span> transport systems&#59; the best known system is represented by the sodium&#47;hydrogen exchanger &#40;NHE&#41; family&#46; Among all the NHE isoforms&#44; NHE isoform 1 &#40;NHE1&#41; is the dominant subtype in hepatic tissue and displays a widespread tissue distribution by maintaining pHi and cell volume levels&#44; in contrast&#44; other subtypes have more limited tissue distribution and are thought to be involved in NaCl absorption&#46;<a class="elsevierStyleCrossRefs" href="#bib0215"><span class="elsevierStyleSup">11&#44;12</span></a></p><p id="par0020" class="elsevierStylePara elsevierViewall">Most studies have demonstrated that apoptosis is ultimately accompanied by cytosol acidification&#46;<a class="elsevierStyleCrossRefs" href="#bib0210"><span class="elsevierStyleSup">10&#44;13&#44;14</span></a> More recently&#44; it was reported that rats subjected to 280<span class="elsevierStyleHsp" style=""></span>mmol&#47;L NH<span class="elsevierStyleInf">4</span>Cl loading for five days developed metabolic acidosis&#46;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">15</span></a> Flow cytometric analysis from our previous research work revealed that the cell apoptosis ratio increased with increasing concentrations of NH<span class="elsevierStyleInf">4</span>Cl&#46; This demonstrated that high blood ammonia levels may lead to liver cell damage and apoptosis&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">4&#8211;6&#44;16</span></a> A cytoprotective role of NHE1 is also supported by experiments in which NHE1-null proximal tubule cells demonstrated enhanced sensitivity to multiple apoptotic stimuli compared with wild-type cells&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">14</span></a> Manucha<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">17</span></a> demonstrated that tubular epithelial cell apoptosis was associated with decreased NHE1 expression in a neonatal rat model of ureteral obstruction&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">In our present study&#44; we investigated whether hyperammonemia has any effects on the pHi and activity of NHE1 in hepatic cells&#46; A hyperammonia hepatic cell model was established to determined the role of NHE1 in the regulation of hepatocyte apoptosis induced by hyperammonaemia and its mechanism underlying liver failure&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Materials</span><p id="par0030" class="elsevierStylePara elsevierViewall">All cell culture reagents&#44; including antibiotics&#44; fetal bovine serum &#40;FBS&#41;&#44; phosphate-buffered saline &#40;PBS&#41; and RIMP1640 were purchased from Thermo Fisher Scientific&#44; Inc&#46; &#40;Waltham&#44; MA&#44; USA&#41;&#46; 2&#8242;&#44;7&#8242;-bis&#40;2-carboxyethyl&#41;-5&#40;6&#41;-carboxyfluo rescein acetoxymethyl ester &#40;BCECF-AM&#41;&#44; cariporide and antibodies were purchased from Santa Cruz Biotechnology&#44; Inc&#46; &#40;Dallas&#44; TX&#44; USA&#41;&#46; Cell culture plastics were purchased from Corning Incorporated &#40;Corning&#44; New York&#44; USA&#41;&#46; Annexin V fluorescein isothiocyanate &#40;FITC&#41; apoptosis detection kit was purchased from Beyotime Institute of Biotechnology &#40;Haimen&#44; China&#41;&#46; All other chemicals were purchased from Sigma&#8211;Aldrich &#40;St&#46; Louis&#44; MO&#44; USA&#41;&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Cell line and experimental treatments</span><p id="par0035" class="elsevierStylePara elsevierViewall">Hepatocytes were obtained from the Type Culture Collection of the Chinese Academy of Sciences&#44; Shanghai&#44; China&#46; When the cells monolayer became confluent&#44; the cells were divided into four groups and incubated for 12<span class="elsevierStyleHsp" style=""></span>h in serum free medium or serum free medium with 10<span class="elsevierStyleHsp" style=""></span>&#956;M cariporide&#44; 5<span class="elsevierStyleHsp" style=""></span>mM NH<span class="elsevierStyleInf">4</span>Cl&#44; or 5<span class="elsevierStyleHsp" style=""></span>mM NH<span class="elsevierStyleInf">4</span>Cl<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleHsp" style=""></span>&#956;M cariporide&#44; respectively&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Intracellular pHi and NHE activity measurement</span><p id="par0040" class="elsevierStylePara elsevierViewall">Hepatocytes were plated on glass coverslips and in complete RIMP1640 at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in a humidified atmosphere with 5&#37; CO<span class="elsevierStyleInf">2</span>&#46; Intracellular pH was measured after chemical treatments&#46; Cellular NHE activity was determined fluorometrically by using intracellular pH-sensitive dye acetoxymethyl ester of BCECF-AM&#44; as described previously&#46;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">10</span></a> In briefly&#44; cells were exposed to Na<span class="elsevierStyleSup">&#43;</span> solution &#40;138<span class="elsevierStyleHsp" style=""></span>mM NaCl&#44; 5<span class="elsevierStyleHsp" style=""></span>mM KCl&#44; 2<span class="elsevierStyleHsp" style=""></span>mM CaCl<span class="elsevierStyleInf">2</span>&#44; 1<span class="elsevierStyleHsp" style=""></span>mM NaH<span class="elsevierStyleInf">2</span>PO<span class="elsevierStyleInf">4</span>&#44; 1<span class="elsevierStyleHsp" style=""></span>mM MgSO<span class="elsevierStyleInf">4</span>&#44; 25<span class="elsevierStyleHsp" style=""></span>mM glucose&#44; and 20<span class="elsevierStyleHsp" style=""></span>mM HEPES&#44; pH 7&#46;4&#41; with 5<span class="elsevierStyleHsp" style=""></span>&#956;M BCECF-AM at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>h&#46; Then cells were washed with Na<span class="elsevierStyleSup">&#43;</span> solution to remove extracellular dye and perfused with Na<span class="elsevierStyleSup">&#43;</span> solution and measured the baseline pHi&#46;</p><p id="par0045" class="elsevierStylePara elsevierViewall">Next&#44; cells were exposed to 40<span class="elsevierStyleHsp" style=""></span>mM NH<span class="elsevierStyleInf">4</span>Cl &#40;pH 7&#46;4&#41; and perfused with Na<span class="elsevierStyleSup">&#43;</span> free medium &#40;130<span class="elsevierStyleHsp" style=""></span>mM tetramethyl ammonium chloride&#44; 5<span class="elsevierStyleHsp" style=""></span>mM KCl&#44; 2<span class="elsevierStyleHsp" style=""></span>mM CaCl<span class="elsevierStyleInf">2</span>&#44; 1<span class="elsevierStyleHsp" style=""></span>mM NaH<span class="elsevierStyleInf">2</span>PO<span class="elsevierStyleInf">4</span>&#44; 1<span class="elsevierStyleHsp" style=""></span>mM MgSO<span class="elsevierStyleInf">4</span>&#44; 25<span class="elsevierStyleHsp" style=""></span>mM glucose&#44; and 20<span class="elsevierStyleHsp" style=""></span>mM HEPES&#44; pH 7&#46;4&#41;&#46; Then&#44; re-addition of extracellular Na<span class="elsevierStyleSup">&#43;</span> allowed activation of Na<span class="elsevierStyleSup">&#43;</span>&#47;H<span class="elsevierStyleSup">&#43;</span> exchange and recovery from acidification to basal levels&#46; At the end of each experiment&#44; the fluorescence ratio was calibrated to pHi by using nigericin &#40;2<span class="elsevierStyleHsp" style=""></span>&#956;M&#41;&#47;high K<span class="elsevierStyleSup">&#43;</span> solutions of different pH values method&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall">Initial rate of Na<span class="elsevierStyleSup">&#43;</span>-dependent recovery from intracellular acidification corresponding to NHE1 activity was almost linear during the first 1<span class="elsevierStyleHsp" style=""></span>min of the reaction&#46; NHE1 activity was measured by calculating the pHi change over the first 1<span class="elsevierStyleHsp" style=""></span>min and expressed as &#916;pH&#47;min&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Cell viability assay</span><p id="par0055" class="elsevierStylePara elsevierViewall">After experimental treatments&#44; cell viability of hepatocytes was demonstrated using the 3-&#40;4&#44;5-dimethylthiazol-2-yl&#41;-2&#44;5-diphenyl-tetrazolium bromide &#40;MTT&#41; assay according to manufacturer&#39;s instructions&#46; In brief&#44; MTT solution &#40;5<span class="elsevierStyleHsp" style=""></span>mg&#47;ml&#41; was added to each well and incubated for 4<span class="elsevierStyleHsp" style=""></span>h at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Then 1<span class="elsevierStyleHsp" style=""></span>ml of dimethyl sulfoxide &#40;DMSO&#41; was added and thoroughly mixed for 10<span class="elsevierStyleHsp" style=""></span>min&#46; Subsequently&#44; MTT absorbance was measured at 570 and 630<span class="elsevierStyleHsp" style=""></span>nm using a microplate reader&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Flow cytometry</span><p id="par0060" class="elsevierStylePara elsevierViewall">To confirm the results from MTT assay&#44; hepatocytes were harvested using trypsin&#47;EDTA solution after experimental treatments&#44; rinsed with binding buffer &#40;0&#46;01<span class="elsevierStyleHsp" style=""></span>M HEPES&#44; 2&#46;5<span class="elsevierStyleHsp" style=""></span>mM CaCl<span class="elsevierStyleInf">2</span>&#44; 0&#46;14<span class="elsevierStyleHsp" style=""></span>M NaCl&#44; pH 7&#46;4&#41; and collected by centrifugation at 1000<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 10<span class="elsevierStyleHsp" style=""></span>min&#46; Cell pellets were resuspended with binding buffer at a concentration of 1<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>cells&#47;ml and incubated with propidium iodide and FITC-Annexin V following the manufacturer&#39;s recommendations for 15<span class="elsevierStyleHsp" style=""></span>min at room temperature in the dark&#46; Cell apoptosis analysis was performed by using a FACScalibur &#40;Becton Dickinson Corp&#46;&#44; USA&#41;&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Measuring intracellular ATP content</span><p id="par0065" class="elsevierStylePara elsevierViewall">Briefly&#44; following cell preparations&#44; intracellular ATP content was measured as described previously<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">18</span></a> by bioluminescence assay using a commercial ATP assay kit &#40;EMD Millipore&#44; Darmstadt&#44; Germany&#41; according to the manufacturer&#39;s instructions&#46; In brief&#44; cells were treated with 100<span class="elsevierStyleHsp" style=""></span>&#956;L nuclear releasing reagent for 5<span class="elsevierStyleHsp" style=""></span>min at room temperature while gently shaking&#44; and 1&#46;0<span class="elsevierStyleHsp" style=""></span>&#956;L ATP monitoring enzyme was added into the cell lysates&#46; Bioluminescence of intracellular ATP content was determined by comparing with a standard curve that was generated by using various ATP solutions with known concentrations&#46; The intracellular ATP is further normalized to protein concentration for comparison&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Western blot analysis</span><p id="par0070" class="elsevierStylePara elsevierViewall">Following experimental treatments&#44; hepatocytes were washed three times by ice-cold PBS containing 50<span class="elsevierStyleHsp" style=""></span>mM Tris&#46; Then hepatocytes were harvested by centrifugation at 750<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 10<span class="elsevierStyleHsp" style=""></span>min at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Cell pellets were resuspended and lysed in 500<span class="elsevierStyleHsp" style=""></span>&#956;l of ice-cold lysis buffer &#40;50<span class="elsevierStyleHsp" style=""></span>mM NaCl&#44; 10<span class="elsevierStyleHsp" style=""></span>mM HEPES&#44; 5<span class="elsevierStyleHsp" style=""></span>mM EDTA&#44; 1<span class="elsevierStyleHsp" style=""></span>mM benzamidine&#44; 0&#46;5&#37; Triton X-100&#41;&#46; Cell lysate was solubilized at 4<span class="elsevierStyleHsp" style=""></span>&#176;C for 30<span class="elsevierStyleHsp" style=""></span>min with end-over-end rotation and subsequently homogenized 10 times using a 26-gauge needle applied to a 1-ml syringe&#46; Cellular debris was cleared by centrifugation at 14&#44;000<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 15<span class="elsevierStyleHsp" style=""></span>min&#46; Supernatant was collected and solubilized in loading buffer &#40;5<span class="elsevierStyleHsp" style=""></span>mM Tris&#8211;HCl&#44; 1&#37; SDS&#44; 10&#37; glycerol&#44; and 1&#37; 2-mercaptoethanol&#44; pH 6&#46;8&#41;&#44; boiled for 10<span class="elsevierStyleHsp" style=""></span>min&#46; Protein content of each fraction was determined by the bicinchoninic acid assay &#40;Sigma&#41;&#46; Subsequently&#44; normalized samples were loaded&#44; size-fractionated by 10&#37; SDS-PAGE&#44; and electrophoretically transferred to nitrocellulose membrane&#46; After blocking with 5&#37; nonfat milk in PBST &#40;0&#46;05&#37; Tween&#47;PBS&#41; at room temperature for 1<span class="elsevierStyleHsp" style=""></span>h and incubated with anti-p-Akt antibody&#44; anti-Akt antibody&#44; anti-p-PI3K antibody and anti-PI3K antibody or anti-&#946;-actin antibody &#40;dilution 1&#58;1000&#41;&#44; respectively&#44; membrane was washed with PBST for three times for 10<span class="elsevierStyleHsp" style=""></span>min each&#46; Following incubation with Alexa Fluor<span class="elsevierStyleSup">&#174;</span>488 mouse anti-rabbit IgG and Alexa Fluor<span class="elsevierStyleSup">&#174;</span> 680 rabbits anti-mouse IgG &#40;Thermo Fisher Scientific&#44; Inc&#46;&#59; dilution 1&#58;10&#44;000&#41; for 60<span class="elsevierStyleHsp" style=""></span>min at room temperature in the dark&#46; Subsequently&#44; lots were detected and relative density units were estimated from the mean pixel density using Image Studio Lite 4&#46;0 and normalized to &#946;-actin&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Statistical analysis</span><p id="par0075" class="elsevierStylePara elsevierViewall">Statistical analyses were performed using SPSS 13&#46;0 software &#40;SPSS&#44; Inc&#46;&#44; Chicago&#44; IL&#44; USA&#41;&#46; Data from multiple experiments were processed and expressed as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; Statistical differences between the different groups were determined using one-way analysis of variance followed by a Student&#8211;Newman&#8211;Keuls test&#46; <span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 was considered to indicate a statistically significant difference&#46;</p></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Results</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">NH<span class="elsevierStyleInf">4</span>Cl and cariporide lowered intracellular pH</span><p id="par0080" class="elsevierStylePara elsevierViewall">To verify the effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on intracellular pH in hepatocytes&#44; the fluorescent indicator dye BCECF-AM was used&#46; <a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a> showed that application of NH<span class="elsevierStyleInf">4</span>Cl produced an obvious intracellular acidification&#46; As expected&#44; cariporide&#44; a specific NHE1 inhibitor&#44; decreased pHi&#44; but its effect on pHi was much less than that in NH<span class="elsevierStyleInf">4</span>Cl medium&#46; However&#44; this intracellular acidification was synergistically increased when they were used together&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on NHE1 Activity</span><p id="par0085" class="elsevierStylePara elsevierViewall">Because NHE activity contributes to the maintenance of intracellular pH homeostasis&#44; we next investigated the effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on NHE1 activity&#46; In hepatic cells&#44; the mean activity of the NHE1 was 0&#46;209<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;005 &#916;pH&#47;min and more than 77&#37; of the sodium dependent pHi recovery is inhibited by cariporide&#46; When hepatic cells were incubated with 5<span class="elsevierStyleHsp" style=""></span>mM NH<span class="elsevierStyleInf">4</span>Cl&#44; Na<span class="elsevierStyleSup">&#43;</span>&#47;H<span class="elsevierStyleSup">&#43;</span> activity significantly increased to 0&#46;261<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;003 &#916;pH&#47;min&#44; whereas addition of cariporide to the culture medium with 5<span class="elsevierStyleHsp" style=""></span>mM NH<span class="elsevierStyleInf">4</span>Cl decreased Na<span class="elsevierStyleSup">&#43;</span>&#47;H<span class="elsevierStyleSup">&#43;</span> activity to the same level as the cariporide group &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on cell viability</span><p id="par0090" class="elsevierStylePara elsevierViewall">The effects of NH<span class="elsevierStyleInf">4</span>Cl on the viability of hepatic cells were valuated by MTT assay&#46; The data revealed that treatment of hepatic cells with cariporide or 5<span class="elsevierStyleHsp" style=""></span>mM NH<span class="elsevierStyleInf">4</span>Cl reduced the cell viability by 10&#46;88&#37; or 12&#46;69&#37; of the control cells&#44; respectively&#46; Whereas cell viability decreased significantly to 68&#46;44&#37; when they were used together &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on cell apoptosis</span><p id="par0095" class="elsevierStylePara elsevierViewall">Because above data suggests good correlation between decreased pHi and increased hepatocytes death&#44; the involvement of apoptosis in the hepatocytes death induced by NH<span class="elsevierStyleInf">4</span>Cl is evaluated by flow cytometry analysis&#46; After 24<span class="elsevierStyleHsp" style=""></span>h treatment with NH<span class="elsevierStyleInf">4</span>Cl or cariporide increased cell apoptosis up to 14&#46;03<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;56&#37; or 16&#46;27<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;38&#37;&#44; respectively&#44; and interestingly&#44; co-incubation with cariporide increased cell apoptosis dramatically in ammonia-treated cells to almost 33&#46;88<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;53&#37; compared with the control &#40;3&#46;16<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;17&#37;&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on cellular ATP</span><p id="par0100" class="elsevierStylePara elsevierViewall">As apoptosis is an ATP-dependent process&#44; we next investigated the effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on cellular ATP&#46; After 24<span class="elsevierStyleHsp" style=""></span>h of treatment with cariporide&#44; cellular ATP in hepatic cells was decreased to 82&#37;&#44; whereas treatment of hepatic cells with 5<span class="elsevierStyleHsp" style=""></span>mM NH<span class="elsevierStyleInf">4</span>Cl reduced cellular ATP to 74&#46;59<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;28&#37;&#44; and addition of cariporide to the culture medium with 5<span class="elsevierStyleHsp" style=""></span>mM NH<span class="elsevierStyleInf">4</span>Cl decreased cellular ATP even more to 55&#37; compared with the control &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>&#41;&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on the ratio of phosphor-Akt &#40;p-Akt&#47;Akt&#41; and phosphor-PI3K &#40;p-PI3K&#47;PI3K&#41;</span><p id="par0105" class="elsevierStylePara elsevierViewall">As the PI3K&#47;Akt pathway is a well-known antiapoptotic pathway&#44; we examined whether NH<span class="elsevierStyleInf">4</span>Cl and cariporide affected the kinase-mediated phosphorylation process in the following experiments&#46; The relative levels of p-Akt&#47;Akt and p-PI3K&#47;PI3K were monitored by western blot analysis&#46; <a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a> shows that cariporide slightly reduced the ratio of p-Akt&#47;Akt and p-PI3K&#47;PI3K&#44; whereas&#44; the ratio of p-Akt&#47;Akt and p-PI3K&#47;PI3K significantly increased in NH<span class="elsevierStyleInf">4</span>Cl treated cells and the upregulated phosphorylation of Akt and PI3K was strongly blocked by cariporide&#46;</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Discussion</span><p id="par0110" class="elsevierStylePara elsevierViewall">The &#8216;secondary injury&#8217; theory of HF indicates that serious metabolic disorders develop&#44; including hyperammonaemia&#44; lactic acid poisoning&#44; blood disorders and increased indole levels&#44; and hyperammonaemia is the most significant causes of liver abnormalities or failure&#46;<a class="elsevierStyleCrossRefs" href="#bib0250"><span class="elsevierStyleSup">18&#44;19</span></a> Many studies supported that hyperammonaemia&#44; which is both the result and the cause of liver damage&#44; may aggravate hepatic damage&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">4&#8211;6&#44;16</span></a></p><p id="par0115" class="elsevierStylePara elsevierViewall">Previous studies from ours showed that hyperammonemia induce liver cell damage&#44; not by inflammation or necrosis&#44; but by cell apoptosis&#46; Cell apoptosis was the predominant effect after treating with low concentration of NH<span class="elsevierStyleInf">4</span>Cl for a short period of time&#46; As the concentration of NH<span class="elsevierStyleInf">4</span>Cl increased and the treatment time lengthened&#44; the number of apoptotic cells increased significantly and the growth of cells was markedly inhibited&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">4&#44;16</span></a></p><p id="par0120" class="elsevierStylePara elsevierViewall">It has been reported that rats subjected to NH<span class="elsevierStyleInf">4</span>Cl loading developed metabolic acidosis<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">15</span></a> and metabolic acidosis increased NHE1 activity&#47;expression&#46;<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">20</span></a> Intracellular pH is a fundamental modulator of protein function in cells&#46; Optimum pH for endonuclease and caspase activation are 6&#46;3&#8211;6&#46;8 and intracellular acidification is critical for apoptosis execution&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">21</span></a> Activation of the NHE1 results in an efflux of H<span class="elsevierStyleSup">&#43;</span> and influx of Na<span class="elsevierStyleSup">&#43;</span> ions with a resultant increase in pHi&#44; and NHE1-induced cytosol alkalinization were sufficient to prevent apoptosis&#46; Additionally&#44; cells undergoing apoptosis exhibit persistent cell shrinkage&#44; even under isotonic environments&#46; NHE1-mediated cytoplasmic volume expansion by inducing Na<span class="elsevierStyleSup">&#43;</span> and H<span class="elsevierStyleInf">2</span>O influx also protect cells against apoptotic volume decreases&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">22</span></a></p><p id="par0125" class="elsevierStylePara elsevierViewall">Cariporide&#44; which is a highly selective NHE-1 inhibitor with no apparent effect on the Na<span class="elsevierStyleSup">&#43;</span>&#47;Ca<span class="elsevierStyleSup">2&#43;</span> exchanger or fast Na<span class="elsevierStyleSup">&#43;</span> currents at levels not exceeding 10<span class="elsevierStyleHsp" style=""></span>&#956;mol&#47;l&#44;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">23</span></a> NHE1 inhibition induced by cariporide has been implicated as an explanation for diminished intracellular pH &#40;pHi&#41;&#44; with possible mechanisms of inactivation including ATP depletion&#44; NHE1 dephosphorylation&#44; or cleavage&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">14</span></a> Cariporide was used in this study because it specifically inhibits the NHE-1 exchanger&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">It has been well known that pHi may contribute to NHE1-mediated modulation of cell proliferation and cells cannot properly differentiate if lacking NHE1&#46;<a class="elsevierStyleCrossRefs" href="#bib0280"><span class="elsevierStyleSup">24&#44;25</span></a> Suppression of apoptosis can be achieved by NHE1 activation and cellular alkalinization&#44; that is stimulated by phosphorylation of the C-terminal regulatory domain of the NHE resulting in increased affinity for intracellular H<span class="elsevierStyleSup">&#43;</span>&#46;<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">26</span></a> In this study&#44; we noted NH<span class="elsevierStyleInf">4</span>Cl treatment caused cytoplasmic acidification&#44; and in response&#44; the activity of NHE1increased&#46; On the contrary&#44; cariporide inhibited the activities of NHE1 and reduced pHi in hepatic cells&#46; And we also noted that the presence of cariporide decreased the activity of NHE-1induced by NH<span class="elsevierStyleInf">4</span>Cl and enhanced cytoplasmic acidification&#46; As expected&#44; we also observed that decreased pHi induced by NH<span class="elsevierStyleInf">4</span>Cl was associated with increased apoptosis and low cell proliferation&#44; which was exacerbated by exposure to the NHE1 inhibitor cariporide&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">It was reported that NHE inhibitors acted as ATP analogs that caused the formation of nonproductive enzyme&#8211;substrate complexes&#46;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">27</span></a> As apoptosis is an ATP-dependent process&#44; we also found the degree of ATP depletion increased when the cells were treated by NH<span class="elsevierStyleInf">4</span>Cl or cariporide&#44; and when they worked together&#44; the degree of ATP depletion became further lower&#46;</p><p id="par0140" class="elsevierStylePara elsevierViewall">All of these findings are consistent with observations that intracellular acidification triggers cell death&#46;<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">20</span></a> NHE1 is likely to be activated in response to decreased pHi and activited NHE1 may then be sufficient to prevent further intracellular acidification and perhaps even promote cell survival&#44; provide the apoptotic stimulus is not too robust&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">21</span></a> Thus it appears that endogenous NHE activity contributes to the maintenance of intracellular pH homeostasis and NHE1 plays a cytoprotective role in mechanics of cell apoptosis&#46; On the contrary&#44; pharmacological inhibition of NHE1 is sufficient to induce or exacerbate apoptotic cell death&#46;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">24</span></a></p><p id="par0145" class="elsevierStylePara elsevierViewall">The PI3K&#47;Akt pathway is a well-known antiapoptotic pathway&#46; Phosphorylated Akt&#44; active form of Akt&#44; activates antiapoptotic proteins such as Bcl-2 and Bcl-xL<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">28</span></a> and prevents cytochrome c leakage from the mitochondria&#46;<a class="elsevierStyleCrossRefs" href="#bib0270"><span class="elsevierStyleSup">22&#44;29</span></a> The present study showed that there is an decrease in phospho-PI3K and phospho-Akt in cariporide medium compared with that in normal medium&#46; On the contrary&#44; ratios of p-Akt&#47;Akt or p-PI3K&#47;PI3K significantly increased under NH<span class="elsevierStyleInf">4</span>Cl treatment and their stimulation were strongly prevented by cariporide treatment&#46; In this context&#44; upregulation of PI3K&#47;Akt pathway activity appears to play a major role in facilitating cell growth and protecting cells from NH<span class="elsevierStyleInf">4</span>Cl-induced apoptosis&#44; but dephosphorylation &#40;inactivation&#41; is responsible for the cariporide -induced enhancement of NH<span class="elsevierStyleInf">4</span>Cl cytotoxicity&#46;</p><p id="par0150" class="elsevierStylePara elsevierViewall">To further clarify the role of the PI3K&#47;Akt pathway linking NHE1 activity&#44; Newell KJ examined NHE1 activity in the presence of three different concentrations of the Akt inhibitor API-2 and found that these inhibitors concentrations had no effect on the rate of acid efflux&#44; thus indicating that Akt is clearly downstream of NHE1&#46; Moreover they also found the decrease in NHE1 activities after cariporide treatment seemed to contribute to reducing ERK phosphorylation&#46;<a class="elsevierStyleCrossRefs" href="#bib0310"><span class="elsevierStyleSup">30&#44;31</span></a> These findings are consistent with that NHE1 uses ERM to bind PIP2 in a signalplex&#44; which induces PI3K expression&#46; PI3K converts PIP2 into phosphatidylinositol &#40;3&#44;4&#44;5&#41;-phosphate &#91;PI&#40;3&#44;4&#44;5&#41; P3&#93; and results in the phosphorylation of Akt&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">22</span></a> Moreover&#44; the mechanism of blocking the kinase-mediated phosphorylaton process was by competing with ATP&#46;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">24</span></a></p><p id="par0155" class="elsevierStylePara elsevierViewall">An important and novel observation in this study was that intracellular acidification induced by NH<span class="elsevierStyleInf">4</span>Cl in hepatic cells&#44; stimulated PI3K and Akt phosphorylation and that this effect was strongly reduced by NHE1 inhibitions&#46; This work has highlighted an important role for NHE1 in the protection of hepatocytes from apoptosis induced by hyperammonaemia&#46; Cariporide is an inhibitor of NHE1 and increases apoptosis it is not clinical beneficious&#44; at least in this condition&#46; So&#44; in medical point of view&#44; it will be interesting to suggest strategies to preserve or increase NHE1 activity that may be therapeutically beneficial for acute liver failure&#46;</p><p id="par0160" class="elsevierStylePara elsevierViewall">Taken together&#44; in light of these findings&#44; we postulate that apoptotic stress triggers NHE1-dependent defense against intracellular acidification&#44; but we should realize that NH4Cl-induced protons leaving the hepatic cells via NHE1 may lead to Na<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>loading&#44; which may subsequently induce Ca2&#43; overloading as Na&#43; leaves the cell via the Na&#43;&#47;Ca2&#43; exchanger if ammonium is cleared out in time&#46; The resultant rise in intracellular calcium concentration is believed to trigger Ca2&#43;-activated protease and phospholipases that cause cellular damage&#46;<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">32</span></a> Those are just speculation&#44; which need further research to confirm&#46; Therefore&#44; NHE1-dependent defense against intracellular acidification plays a role in the protective effect in the early stages of apoptosis induced by hyperammonaemia&#46;</p></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Conclusion</span><p id="par0165" class="elsevierStylePara elsevierViewall">Increased levels of ammonia in the blood may be an important cause of further damage to residual liver cells following liver failure&#46; Intracellular acidification induced by NH4Cl triggers cell death and cell apoptosis was the predominant effect in the early injury of liver cells&#44; induced by high levels of blood ammonia&#46; The injury of hepatocytes induced by high ammonia may be related to the reduction of pHi&#44; and increased activation of NHE1 protein in hepatocytes via an increase in phosphorylated Akt to suppress hepatocytes apoptosis&#44; while cariporide inhibits NHE1 activities and amplifies the intracellular acidosis and consequently enhance NH4Cl-induced cytotoxicity via diminution of Akt phosphorylation&#44; verified that suppression of apoptosis can be achieved by NHE1 activation and cellular alkalinization&#46; Overall&#44; our model may provide important insights into how NHE1 plays a role in the protective effect in the early stages of apoptosis induced by hyperammonaemia and we believe that this model provides a framework for future studies&#46;</p></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0150">Conflicts of interest</span><p id="par0170" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest&#46;</p></span></span>"
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              "titulo" => "Cell line and experimental treatments"
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              "titulo" => "Intracellular pHi and NHE activity measurement"
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              "titulo" => "Cell viability assay"
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              "titulo" => "Flow cytometry"
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              "titulo" => "Measuring intracellular ATP content"
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              "titulo" => "Western blot analysis"
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          "titulo" => "Results"
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              "titulo" => "NHCl and cariporide lowered intracellular pH"
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            1 => array:2 [
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              "titulo" => "Effects of NHCl and cariporide on NHE1 Activity"
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              "identificador" => "sec0070"
              "titulo" => "Effects of NHCl and cariporide on cell viability"
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              "identificador" => "sec0075"
              "titulo" => "Effects of NHCl and cariporide on cell apoptosis"
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              "identificador" => "sec0080"
              "titulo" => "Effects of NHCl and cariporide on cellular ATP"
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            5 => array:2 [
              "identificador" => "sec0085"
              "titulo" => "Effects of NHCl and cariporide on the ratio of phosphor-Akt &#40;p-Akt&#47;Akt&#41; and phosphor-PI3K &#40;p-PI3K&#47;PI3K&#41;"
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          "identificador" => "xack370049"
          "titulo" => "Acknowledgements"
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          "titulo" => "References"
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    "fechaRecibido" => "2017-10-10"
    "fechaAceptado" => "2018-05-24"
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          "clase" => "keyword"
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          "palabras" => array:4 [
            0 => "Hyperammonaemia"
            1 => "Intracellular pH"
            2 => "Sodium-hydrogen exchanger isoform 1"
            3 => "Apoptosis"
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          "palabras" => array:4 [
            0 => "Hiperamoniaquemia"
            1 => "pH intracelular"
            2 => "Isoforma 1 del intercambiador i&#243;nico de sodio&#47;hidr&#243;geno"
            3 => "Apoptosis"
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        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Background</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">The &#8220;secondary injury&#8221; theory of liver failure indicated that hyperammonaemia due to liver failure causes further deterioration of hepatocytes&#46; Our previous studies have demonstrated that high blood ammonia levels may lead to hepatocyte apoptosis&#44; as NH<span class="elsevierStyleInf">4</span>Cl loading caused metabolic acidosis and an increase in sodium-hydrogen exchanger isoform 1 &#40;NHE1&#41;&#46; In this study&#44; we established a hyperammonia hepatocyte model to determine the role of NHE1 in the regulation of hepatocyte apoptosis induced by NH<span class="elsevierStyleInf">4</span>Cl&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Materials and methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">In current studies&#44; intracellular pH &#40;pHi&#41; and NHE1 activity were analyzed using the pHi-sensitive dye BCECF-AM&#46; The results showed that intracellular pH dropped and NHE1 activity increased in hepatocytes under NH<span class="elsevierStyleInf">4</span>Cl treatment&#46; As expected&#44; decreased pHi induced by NH<span class="elsevierStyleInf">4</span>Cl was associated with increased apoptosis&#44; low cell proliferation and ATP depletion&#44; which was exacerbated by exposure to the NHE1 inhibitor cariporide&#46; We also found that NH<span class="elsevierStyleInf">4</span>Cl treatment stimulated PI3K and Akt phosphorylation and this effect was considerably reduced by NHE1 inhibition&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Conclusion</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">This study highlighted the significant role of NHE1 in the regulation of cell apoptosis induced by hyperammonaemia&#46;</p></span>"
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          0 => array:2 [
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        "resumen" => "<span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Antecedentes</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">La teor&#237;a de la &#171;lesi&#243;n secundaria&#187; de la insuficiencia hep&#225;tica mostr&#243; que la hiperamoniaquemia provocada por la insuficiencia hep&#225;tica causa mayor deterioro de los hepatocitos&#46; Nuestros anteriores estudios previos han demostrado que los niveles altos de amon&#237;aco en sangre pueden conducir a la apoptosis de los hepatocitos&#46; Como la carga de NH<span class="elsevierStyleInf">4</span>Cl provoc&#243; acidosis metab&#243;lica y un aumento de la isoforma 1 del intercambiador de sodio&#47;hidr&#243;geno &#40;NHE1&#41;&#46; En este estudio&#44; establecimos un modelo de hepatocitos de hiperamonia para establecer el papel de NHE1 en la regulaci&#243;n de la apoptosis de hepatocitos inducida por NH<span class="elsevierStyleInf">4</span>Cl&#46;</p></span> <span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Materiales y m&#233;todos</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">En los estudios actuales&#44; el pH intracelular &#40;pHi&#41; y la actividad del NHE1 se analizaron con el colorante BCECF-AM&#44; sensible al pHi&#46; Los resultados mostraron que el pH intracelular disminuy&#243; y la actividad del NHE1 aument&#243; en hepatocitos con tratamiento del NH<span class="elsevierStyleInf">4</span>Cl&#46; Como se esperaba&#44; la disminuci&#243;n del pHi inducido por NH<span class="elsevierStyleInf">4</span>Cl se relacion&#243; con un aumento de la apoptosis&#44; baja proliferaci&#243;n celular y reducci&#243;n del ATP&#44; que se exacerb&#243; por la exposici&#243;n a cariporide&#44; inhibidor del NHE1&#46; Tambi&#233;n encontramos que el tratamiento del NH<span class="elsevierStyleInf">4</span>Cl estimul&#243; la fosforilaci&#243;n de PI3K y Akt&#44; y este efecto se redujo considerablemente por la inhibici&#243;n del NHE1&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Conclusi&#243;n</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Este trabajo ha destacado el importante papel del NHE1 en la regulaci&#243;n de la apoptosis celular inducida por hiperamoniaquemia&#46;</p></span>"
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          "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Intracellular acidification upon treatment with NH<span class="elsevierStyleInf">4</span>Cl and cariporide &#40;x&#175;&#177;sd&#44; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>9&#41;&#46; Hepatic cells were treated for 24<span class="elsevierStyleHsp" style=""></span>h with NH<span class="elsevierStyleInf">4</span>Cl&#44; cariporide or NH<span class="elsevierStyleInf">4</span>Cl plus cariporide&#46; Control cells were untreated&#46; The intracellular pH &#40;pHi&#41; was then determined with the use of BCECF-AM as described under &#8220;Materials and methods&#8221;&#46; Results are presented as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation of three independent experiments and samples were analyzed in triplicate&#46;</p>"
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          "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on NHE1 Activity &#40;x&#175;&#177;sd&#44; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>9&#41;&#46; Hepatic cells were treated for 24<span class="elsevierStyleHsp" style=""></span>h with NH<span class="elsevierStyleInf">4</span>Cl&#44; cariporide or NH<span class="elsevierStyleInf">4</span>Cl plus cariporide&#44; respectively&#46; Control cells were untreated&#46; NHE activity was measured by calculating the pHi change over the first 1<span class="elsevierStyleHsp" style=""></span>min and expressed as &#916;pH&#47;min with the use of BCECF-AM as described under &#8220;Materials and methods&#8221;&#46; Results are presented as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation of three independent experiments and samples were analyzed in triplicate&#46;</p>"
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Cell viability upon treatment with NH<span class="elsevierStyleInf">4</span>Cl and cariporide &#40;x&#175;&#177;sd&#44; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>9&#41;&#46; Hepatic cells were treated for 24<span class="elsevierStyleHsp" style=""></span>h with NH<span class="elsevierStyleInf">4</span>Cl&#44; cariporide or NH<span class="elsevierStyleInf">4</span>Cl plus cariporide&#44; respectively&#46; Control cells were untreated&#46; Cell viability was determined by MTT assay as described under &#8220;Materials and methods&#8221;&#44; and was presented as optical density versus control&#46; Results are presented as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation of three independent experiments and samples were analyzed in triplicate&#46;</p>"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Cell apoptosis upon treatment with NH<span class="elsevierStyleInf">4</span>Cl and cariporide &#40;x&#175;&#177;sd&#44; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>9&#41;&#46; Hepatic cells were treated for 24<span class="elsevierStyleHsp" style=""></span>h with NH<span class="elsevierStyleInf">4</span>Cl&#44; cariporide or NH<span class="elsevierStyleInf">4</span>Cl plus cariporide&#44; respectively&#46; Control cells were untreated&#46; Cell injury was determined as described under &#8220;Materials and methods&#8221;&#44; and was presented as the percentage of cells with FITC-Annexin V binding excluding propidium iodide staining in total cell numbers&#46; Results are presented as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation of three independent experiments and samples were analyzed in triplicate&#46;</p>"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on cellular ATP &#40;x&#175;&#177;sd&#44; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>9&#41;&#46; Hepatic cells were treated for 24<span class="elsevierStyleHsp" style=""></span>h with NH<span class="elsevierStyleInf">4</span>Cl&#44; cariporide or NH<span class="elsevierStyleInf">4</span>Cl plus cariporide&#44; respectively&#46; Control cells were untreated&#46; Cellular ATP was determined as described under &#8220;Materials and methods&#8221;&#46; Values are presented as the percentage vs&#46; control&#46; Results are presented as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation of three independent experiments and samples were analyzed in triplicate&#46;</p>"
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          "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">Effects of NH<span class="elsevierStyleInf">4</span>Cl and cariporide on p-Akt&#47;Akt and p-PI3K&#47;PI3K &#40;x&#175;&#177;sd&#44; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>3&#41;&#46; Hepatic cells were treated for 24<span class="elsevierStyleHsp" style=""></span>h with NH<span class="elsevierStyleInf">4</span>Cl&#44; cariporide or NH<span class="elsevierStyleInf">4</span>Cl plus cariporide&#44; respectively&#46; Control cells were untreated&#46; Normalized samples were determined by western blot analysis using &#946;-actin as the loading control&#46; Values were presented as ratios of p-Akt&#47;Akt or p-PI3K&#47;PI3K&#46; Results are presented as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation of three independent experiments&#46;</p>"
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    "bibliografia" => array:2 [
      "titulo" => "References"
      "seccion" => array:1 [
        0 => array:2 [
          "identificador" => "bibs0015"
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              "etiqueta" => "1"
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                0 => array:2 [
                  "contribucion" => array:1 [
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