NHE3 Expression Research Articles

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

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

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

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

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

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

Gene expression profiling of proximal and distal renal tubules in Atlantic salmon (Salmo salar) acclimated to fresh water and seawater.

Euryhaline teleost kidneys undergo a major functional switch from being filtratory in freshwater (FW) to being predominantly secretory in seawater (SW) conditions. The transition involves both vascular and tubular effects. There is consensus that the glomerular filtration rate is greatly reduced upon exposure to hyperosmotic conditions. Yet, regulation at the tubular level has only been examined sporadically in a few different species. This study aimed to obtain a broader understanding of transcriptional regulation in proximal versus distal tubular segments during osmotic transitions. Proximal and distal tubule cells were dissected separately by laser capture microdissection, RNA was extracted, and relative mRNA expression levels of >30 targets involved in solute and water transport were quantified by quantitative PCR in relation to segment type in fish acclimated to FW or SW. The gene categories were aquaporins, solute transporters, fxyd proteins, and tight junction proteins. aqp8bb1, aqp10b1, nhe3, sglt1, slc41a1, cnnm3, fxyd12a, cldn3b, cldn10b, cldn15a, and cldn12 were expressed at a higher level in proximal compared with distal tubules. aqp1aa, aqp1ab, nka-a1a, nka-a1b, nkcc1a, nkcc2, ncc, clc-k, slc26a6C, sglt2, fxyd2, cldn3a, and occln were expressed at a higher level in distal compared with proximal tubules. Expression of aqp1aa, aqp3a1, aqp10b1, ncc, nhe3, cftr, sglt1, slc41a1, fxyd12a, cldn3a, cldn3b, cldn3c, cldn10b, cldn10e, cldn28a, and cldn30c was higher in SW- than in FW-acclimated salmon, whereas the opposite was the case for aqp1ab, slc26a6C, and fxyd2. The data show distinct segmental distribution of transport genes and a significant regulation of tubular transcripts when kidney function is modulated during salinity transitions.

Altered absorptive function in the gall bladder during cholesterol gallstone formation is associated with abnormal NHE3 complex formation.

Dysfunction of the Na+/H+ exchanger 3 (NHE3) contributes to the formation of cholesterol gallstones. We aimed to investigate whether NHE3 dysfunction is associated with abnormalities in NHE3 complex formation. We fed C57BL/6 mice with control or lithogenic diet and study the expression of NHE3, ezrin, and Na+/H+ exchanger regulatory factor 1 (NHERF1) in the gallbladder (GB) using RT-PCR and western blot. Immunofluorescence and immunoprecipitation were performed to investigate the interactions of NHE3 with ezrin or NHERF1. To explore the initiating factor that leads to NHE3 dysfunction, we stimulated cholangiocarcinoma cells with taurochenodeoxycholate (TCDC) and/or forskolin. The effects of TCDC on the expression of NHE3 regulatory proteins, as well as their bindings to NHE3, were detected by western blot and immunoprecipitation. Enzyme-linked immunosorbent assay was used to study the regulation of cAMP production by TCDC. The expression of NHERF1 and ezrin phosphorylation level were increased in the gallbladder epithelial cells (GBECs) of C57BL/6 mice with cholesterol gallstones. Immunofluorescence studies demonstrated that the subcellular localization of ezrin and NHERF1 were similar to that of NHE3 in GBECs. Immunoprecipitation revealed that ezrin formed macrocomplex with NHE3, which were enhanced after gallstone formation. TCDC increased forskolin-induced cAMP accumulation, and NHERF1 and PKCα expression in cholangiocarcinoma cells. Under the synergistic effect of forskolin, TCDC stimulated ezrin phosphorylation, with enhanced interaction between ezrin and NHE3. The formation of cholesterol gallstones is associated with abnormal formation of NHE3 complexes. Decreased biliary TCDC may be an initiating factor that leads to abnormal GB absorption.

P0634RISK OF HYDROELECTROLYTIC AND ACID-BASE DISORDERS INDUCED BY LIPOSOMAL AMPHOTERICIN B USE IN VISCERAL LEISHMANIASIS PATIENTS

Abstract Background and Aims Visceral leishmaniasis (VL) is a serious illness if untreated. Liposomal Amphotericin B (L-AMB) is the more effectiveness treatment against LV and less nephrotoxic formulation. We aim to evaluate expression of kidney transporters in VL patients before and during L-AMB use. Method This is a prospective study with 9 selected VL patients that used only L-AMB during hospital stay. Renal transporter analyzes were from before and during the treatment and compared with a control group composed by healthy people. The urine transporters (AQP2, aquaporin 2; NKCC2, Na-K-2Cl cotransporter; NHE3, Na/H exchanger) and a constitutive exosome marker (TSG 101) expressions were measured after urinary exosomes isolation. Results No significant increase of TSG 101 was observed in all comparisons, including between both times and vs healthy controls. Additionally, were observed increased urinary protein expression in VL patients in both times (before and after L-AMB use) in comparison with healthy controls of the AQP2: (105.32 (22.90 – 140.27) vs 1362.97 (627.93-2620.09) or 846,20(552.60-1249.14) %, p<0.01); NHE3: (74.81 (57.17 – 126.33) vs 3373.36 (282.69-6877.97) or 462.67 (123.80-7280.21) %, p<0.01); and NKCC2: (90.98 (79.07 – 120.30) vs 634.45 (345.18-1588.41) or 545.74 (284.34-2183.87) %, p=0.03). Moreover, the patients treated with L-AMB showed a significant decrease in AQT2 (P=0.023) and NHE3 (P=0.008) expression. Conclusion We suggest that despite less nephrotoxic, the L-AMB use may be monitored to investigate patients at risk of hydroelectrolytic and acid-base disorders.

Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake.

Disruption of blood-brain barrier integrity and dramatic failure of brain ion homeostasis including fluctuations of pH occurs during cortical spreading depression (CSD) events associated with several neurological disorders, including migraine with aura, traumatic brain injury and stroke. NHE1 is the primary regulator of pH in the central nervous system. The goal of the current study was to investigate the role of sodium-hydrogen exchanger type 1 (NHE1) in blood brain barrier (BBB) integrity during CSD events and the contributions of this antiporter on xenobiotic uptake. Using immortalized cell lines, pharmacologic inhibition and genetic knockdown of NHE1 mitigated the paracellular uptake of radiolabeled sucrose implicating functional NHE1 in BBB maintenance. In contrast, loss of functional NHE1 in endothelial cells facilitated uptake of the anti-migraine therapeutic, sumatriptan. In female rats, cortical KCl but not aCSF selectively reduced total expression of NHE1 in cortex and PAG but increased expression in trigeminal ganglia; no changes were seen in trigeminal nucleus caudalis. Thus, in vitro observations may have a significance in vivo to increase brain sumatriptan levels. Pharmacological inhibition of NHE1 prior to cortical manipulations enhanced the efficacy of sumatriptan at early time-points but induced facial sensitivity alone. Overall, our results suggest that dysregulation of NHE1 contributes to breaches in BBB integrity, drug penetrance, and the behavioral sensitivity to the antimigraine agent, sumatriptan.

TGFβ Signaling Increases Net Acid Extrusion, Proliferation and Invasion in Panc-1 Pancreatic Cancer Cells: SMAD4 Dependence and Link to Merlin/NF2 Signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death, with a 5-year survival of <10% and severely limited treatment options. PDAC hallmarks include profound metabolic acid production and aggressive local proliferation and invasiveness. This phenotype is supported by upregulated net acid extrusion and epithelial-to-mesenchymal transition (EMT), the latter typically induced by aberrant transforming growth factor-β (TGFβ) signaling. It is, however, unknown whether TGFβ-induced EMT and upregulation of acid extrusion are causally related. Here, we show that mRNA and protein expression of the net acid extruding transporters Na+/H+ exchanger 1 (NHE1, SLC9A1) and Na+, HCO cotransporter 1 (NBCn1, SLC4A7) are increased in a panel of human PDAC cell lines compared to immortalized human pancreatic ductal epithelial (HPDE) cells. Treatment of Panc-1 cells (which express SMAD4, required for canonical TGFβ signaling) with TGFβ-1 for 48 h elicited classical EMT with down- and upregulation of epithelial and mesenchymal markers, respectively, in a manner inhibited by SMAD4 knockdown. Accordingly, less pronounced EMT was induced in BxPC-3 cells, which do not express SMAD4. TGFβ-1 treatment elicited a SMAD4-dependent increase in NHE1 expression, and a smaller, SMAD4-independent increase in NBCn1 in Panc-1 cells. Consistent with this, TGFβ-1 treatment led to elevated intracellular pH and increased net acid extrusion capacity in Panc-1 cells, but not in BxPC-3 cells, in an NHE1-dependent manner. Proliferation was increased in Panc-1 cells and decreased in BxPC-3 cells, upon TGFβ-1 treatment, and this, as well as EMT per se, was unaffected by NHE1- or NBCn1 inhibition. TGFβ-1-induced EMT was associated with a 4-fold increase in Panc-1 cell invasiveness, which further increased ~10-fold upon knockdown of the tumor suppressor Merlin (Neurofibromatosis type 2). Knockdown of NHE1 or NBCn1 abolished Merlin-induced invasiveness, but not that induced by TGFβ-1 alone. In conclusion, NHE1 and NBCn1 expression and NHE-dependent acid extrusion are upregulated during TGFβ-1-induced EMT of Panc-1 cells. NHE1 upregulation is SMAD4-dependent, and SMAD4-deficient BxPC-3 cells show no change in pHi regulation. NHE1 and NBCn1 are not required for EMT per se or EMT-associated proliferation changes, but are essential for the potentiation of invasiveness induced by Merlin knockdown.

Sex Difference In Kidney Electrolyte Transport III: Impact of Low K intake on Thiazide‐Sensitive Cation Excretion in Male and Female Mice

The thiazide sensitive Na‐Cl co‐transporter (NCC) in the distal tubule can be regulated by dietary potassium intake and is critical for electrolyte homeostasis. Previously we reported that there was higher NCC activity and expression in female over male mice and high‐K intake preserved the sex‐difference of NCC‐mediated sodium absorption due to comparable reductions of NCC activity and expression in both sexes. However, with high‐K intake hydrochlorothiazide (HCTZ)‐induced kaliuresis was greater in males than in female mice, suggesting a more powerful male adaptation to high‐K intake. In this study, we explored the role of NCC in adaptation to a low‐K (LK) diet. We measured HCTZ‐induced changes in urine volume (UV), glomerular filtration rate (GFR), absolute (ENa, EK) and fractional (FENa, FEK) cation excretion in male and female mice on control‐K (CK, 1% KCl) and LK (0.1%, KCl) diets for 7 days. We also examined LK‐induced changes in NCC, Na/H‐exchanger isoform 3 (NHE3), and ENaC protein expression by Western blotting. With CK, NCC‐dependent ENa and FENa were larger in females than males as we observed previously. However with LK HCTZ‐induced ENa and FENa increased in males but not in females. Thus K restriction abolished the sex differences in NCC function. Changes in total NCC protein followed a similar pattern. Under CK conditions females expressed more NCC protein, as previously reported. LK increased NCC abundance in both males (by 100%) and females (by 40%). The larger effect in males reversed the sex‐dependence of NCC expression, consistent with the measurements of function. LK intake did not change either NHE3 or NHE2 expression, but reduced the amount of cleaved (presumably active) forms of α and γENaC expression in males but not in females. Thus decreased Na reabsorption through ENaC may compensate for increased Na transport through NCC in males. Despite large diuretic and natriuretic responses to HCTZ, EK was only slightly increased in response to the drug when animals were on LK. This suggests that the K‐secretory apparatus in the distal nephron is strongly suppressed under these conditions.Support or Funding InformationNIH NIDDK RO1 DK099284

Proximal tubule albumin uptake – potential role for endothelin system in sickle cell disease mice

Elevated plasma endothelin‐1 (ET‐1) reported in sickle cell disease (SCD) patients correlate with microalbuminuria, a major mortality risk factor in SCD. ET‐1 contributes to glomerular and tubular injury in SCD, as evidenced by increased glomerular permeability to albumin and urinary excretion of tubular injury markers. Although selective ETA receptor antagonism reduces albuminuria in humanized sickle cell (HbSS) mice, the mechanism of this action remains unclear. The aim of the study was to determine whether selective ETA receptor antagonism prevents albuminuria by preserving the expression of tubular albumin‐associated transporters in proximal tubule (PT) cells. Male C57BL/6 or HbSS and genetic control (HbAA) mice were used to determine the effect of ET‐1 on the expression of proximal tubule albumin trafficking mediators, such as megalin and NHE‐3. Exposure of primary mouse PT cells to ET‐1 (50nM) for 8h decreased megalin (53% reduction) and doubled NHE‐3 expression. Pre‐treatment with the ETA antagonist, BQ123 (1 μM), preserved megalin expression and had no effect on NHE‐3. Moreover, selective ETB receptor blockade (BQ788, 1 μM) prevented ET‐1‐mediated increase in NHE‐3 expression. Primary PT cells isolated from HbSS mice showed decreased megalin mRNA expression as well as protein abundance relative to HbAA PT controls. Ten‐week treatment with selective ETA receptor antagonist (10mg/kg/day) preserved expression of megalin at control levels. There were no differences in NHE‐3 mRNA expression in HbSS PT cells regardless the treatment. Interestingly, PT cells from HbSS cultured with HbSS plasma and ET‐1 (10nM) had decreased NHE‐3 protein abundance compared to non‐treated cells. These results potentially uncover a novel role for ET‐1 in PT albumin handling and suggest that PT ET‐1 receptor signaling contributes to albuminuria in SCD.Support or Funding InformationSupport provided by an NIH U01 HL117684 to D.M.P, AHA 19CDA34660073 and NIH‐NHLBI K99HL144817 to M.K.

LAMC2 modulates the acidity of microenvironments to promote invasion and migration of pancreatic cancer cells via regulating AKT-dependent NHE1 activity

LAMC2, as a unique chain in the Laminin 5 molecule, has been found to be associated with malignant metastases in some cancers. However, the roles and mechanisms by which LAMC2 affects the migration and invasion of pancreatic cancer cells remain unclear. First, we found that laminin 5/LAMC2 and its receptors were highly expressed in pancreatic cancer tissues and cells. Then, we investigated the effects of LAMC2 on pancreatic cancer cell migration/invasion and extracellular (pHe). We also demonstrated that LAMC2 phosphorylated Akt-Ser473 to promote the expression, activity and cell membrane accumulation of NHE1 within pancreatic cancer cells. So we speculated that LAMC2 modulated the pHe to promote migration and invasion of pancreatic cancer cells. Additionally, our data also showed that LAMC2/NHE1 resulted in altered cell morphology and aberrant expression of mesenchymal markers. The function of actin-binding proteins (ABPs) were affected by LAMC2/NHE1 signaling. LAMC2/NHE1 signaling generated extracellular acidification to induce dynamic actin-dependent pseudopodial formation and EMT programs that promote tumor cell invasion in pancreatic cancer cells. Therefore, we found that LAMC2 was responsible for generating the extracellular acidic conditions that mediated invasion of pancreatic cancer cells by activating Akt/NHE1 signaling. LAMC2 is a characteristic prognostic and therapeutic agent of PDCA.

Role of PCSK1N and GPR83 in salt resistance

Previous studies from our laboratory show that in mice increased expression/function of the orphan receptor GPR83 is associated with salt resistance. GPR83 is expressed in proximal and distal convoluted tubules of the mouse kidney. Global deletion or renal‐specific silencing of the GPR83 gene increases systolic blood pressure (SBP) and increased G protein‐coupled receptor kinase type 4 (GRK4) expression in the kidney. Recent evidence indicated that peptides derived from the Proprotein Convertase Subtilisin/Kexin Type 1 Inhibitor (PCSK1N) may be the endogenous ligands of GPR83. We hypothesized that these peptides are implicated in the regulation of blood pressure. Renal specific siRNA‐mediated silencing of PCSK1N in mice resulted in an increase in SBP (130±4 vs control wild‐type 100±1 mmHg, n=3/group; P&lt;0.05); this was associated with decreased urinary Na+ excretion (P&lt;0.05) and decreased renal cortex expression of GPR83, but increased NHE3 and Na+/K+‐ATPase expressions (P&lt;0.05 all). The peptides PEN (proline, glutamic acid, and asparagine) and SAAS (serine, alanine, alanine, and serine) are derived from enzymatic breakdown of Pro‐SAAS. Renal restricted infusion of PEN (1 μg/day; 7 days) did not affect SBP, slightly decreased Na+ excretion and increased GPR83, GRK4, and NHE3 expressions (P&lt;0.05 all) but did not affect Na+/K+‐ATPase expression. By contrast, renal‐restricted infusion of SAAS (1 μg/day; 7 days) in mice decreased SBP (82± vs 100± mmHg; P&lt;0.05), Na+ excretion did not change but decreased the renal cortex expression of GRK4, GPR83, NHE3 and Na+/K+‐ATPAse (P&lt;0.05 all). Furthermore, PCSK1N expression was increased by a high salt diet (4%NaCl) in non‐transgenic mice but was decreased in the salt‐sensitive GRK4 486V mice on the same diet. Our result suggested that Pro‐SAAS peptides may be involved in the development of salt sensitivity through gene‐gene interactions among PCSK1N, GPR83, and GRK4.Support or Funding InformationThis work was supported by NIH grant t R01DK039308 (P.A.J.);NIH grant R37HL023081(P.A.J.); NIH grant P01HL074940 (P.A.J.); NIH grant P01HL068686 (P.A.J.); NIH grant R01HL092196 (P.A.J., I.A.); NIH grant R01DK09098 (I.A.)

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

Decreased NHE3 activity and trafficking in TGEV-infected IPEC-J2 cells via the SGLT1-mediated P38 MAPK/AKt2 pathway

Transmissible gastroenteritis virus (TGEV) primarily replicates in intestinal epithelial cells and causes severe damage to host cells, resulting in diarrhea. Surface NHE3 serves as the key regulatory site controlling electroneutral Na+ absorption. In this study, our results showed that the surface NHE3 content was significantly reduced following TGEV infection, whereas the total level of protein expression was not significantly changed, and NHE3 activity gradually decreased with prolonged infection time. We then inhibited SGLT1 expression by lentiviral interference and drug inhibition, respectively. Inhibition studies showed that the level of phosphorylation of the downstream key proteins, MAPKAPK-2 and EZRIN, in the SGLT1-mediated p38MAPK/AKt2 signaling pathway was significantly increased. The surface NHE3 expression was also significantly increased, and NHE3 activity was also significantly enhanced. These results demonstrate that a TGEV infection can inhibit NHE3 translocation and attenuates sodium-hydrogen exchange activity via the SGLT1-mediated p38MAPK/AKt2 signaling pathway, affecting cellular electrolyte absorption leading to diarrhea.

Salt-water acclimation of the estuarine crocodile Crocodylus porosus involves enhanced ion transport properties of the urodaeum and rectum.

Estuarine crocodiles, Crocodylus porosus, inhabit freshwater, estuarine and marine environments. Despite being known to undertake extensive movements throughout and between hypo-osmotic and hyperosmotic environments, little is known about the role of the cloaca in coping with changes in salinity. We report here that, in addition to the well-documented functional plasticity of the lingual salt glands, the middle of the three cloacal segments (i.e. the urodaeum) responds to increased ambient salinity to enhance solute-coupled water absorption. This post-renal modification of urine serves to conserve water when exposed to hyperosmotic environments and, in conjunction with lingual salt gland secretions, enables C. porosus to maintain salt and water balance and thereby thrive in hyperosmotic environments. Isolated epithelia from the urodaeum of 70% seawater-acclimated C. porosus had a strongly enhanced short-circuit current (an indicator of active ion transport) compared with freshwater-acclimated crocodiles. This enhanced active ion absorption was driven by increased Na+/K+-ATPase activity, and possibly enhanced proton pump activity, and was facilitated by the apical epithelial Na+ channel (ENaC) and/or the apical Na+/H+ exchanger (NHE2), both of which are expressed in the urodaeum. NHE3 was expressed at very low levels in the urodaeum and probably does not contribute to solute-coupled water absorption in this cloacal segment. As C. porosus does not appear to drink water of salinities above 18ppt, observations of elevated short-circuit current in the rectum as well as a trend for increased NHE2 expression in the oesophagus, the anterior intestine and the rectum suggest that dietary salt intake may stimulate salt and possibly water absorption by the gastrointestinal tract of C. porosus living in hyperosmotic environments.

Ion Transport Basis of Diarrhea in a Mouse Model of Adoptive T Cell Transfer Colitis.

Diarrhea, a major pathological hallmark of inflammatory bowel disease, is characterized by a significant reduction in the expression and function of key intestinal ion transporters. The adoptive naïve CD4+ T cell transfer colitis is an immune-based, chronic colitis mouse model which resembles human Crohn's disease. Although mice with T cell transfer colitis demonstrate diarrhea, the ion transporter basis of this phenotype has not been explored. In the current studies, we aimed to determine the mRNA and protein levels of the key NaCl transporters DRA and NHE3 along with the mRNA expression of other transporters in the inflamed intestine. Naïve CD4+ T cells, transferred to Rag2 knockout mice, induced severe colonic inflammation characterized by histological damage and increased mRNA levels of cytokines in the colon with no effect in the ileum. Diarrheal phenotype was a key feature of the excised colons of mice where loose stools were evident. Our results demonstrated that the key chloride transporter DRA, mRNA, and protein levels were significantly reduced in the inflamed colon. However, expression of the key sodium hydrogen exchanger NHE3 was unaffected. The mRNA expression of other important transporters was also determined; in this regard, the sodium channel ENACα and the monocarboxylate transporters MCT1 and SMCT1 mRNA levels were also significantly lower compared to control mice. However, CFTR mRNA was not altered in the colon or ileum. The studies conducted herein for the first time demonstrate the downregulation of important intestinal ion transporters in proximal and distal colon in T cell transfer colitis mouse model, providing valuable evidence for the ion transporter basis of diarrhea in this chronic model of inflammation.

MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells

BackgroundBoth cell adhesion and matrix metalloproteinase (MMP) activity depend on pH at the cell surface. By regulating extracellular juxtamembrane pH, the Na+/H+ exchanger NHE1 plays a significant part in human melanoma (MV3) cell migration and invasion. Because NHE1, besides its pH-regulatory transport function, also serves as a structural element tying the cortical actin cytoskeleton to the plasma membrane, we investigated whether NHE1 affects cortical stiffness of MV3 cells, and how this makes an impact on their invasiveness.MethodsNHE1 overexpressing MV3 cells were compared to the corresponding mock-transfected control cells. NHE1 expression was verified by Western blotting, cariporide (HOE642) was used to inhibit NHE1 activity, cell stiffness was determined by atomic force microscopy, and F-actin was visualized by phalloidin-staining. Migration on, and invasion of, native and glutaraldehyde-fixed collagen I substrates were analyzed using time-lapse video microscopy and Boyden-chamber assays, respectively. MMP secretion and activity were detected by Western blot and zymography, respectively. MMP activity was inhibited with NNGH.ResultsThe cortical, but not the bulk stiffness, was significantly higher in NHE1 overexpressing cells. This increase in cortical stiffness was accompanied by a reorganization of the cortical cytoskeleton, i.e. a condensation of F-actin underneath and along the plasma membrane. However, it was not affected by NHE1 inhibition. Nevertheless, actin dynamics is required for cell invasion as demonstrated with the application of cytochalasin D. NHE1 overexpression was associated with an elevated MMP3 secretion and an increase in the invasion of a native matrix. This increase in invasiveness could be antagonized by the MMP inhibitor NNGH. Transmigration through a glutaraldehyde-fixed, indigestible substrate was not affected by NHE1 overexpression.ConclusionNHE1, as a structural element and independently of its transport activity, contributes to the organization of the cortical F-actin meshwork and thus impacts cortical stiffness. Since NHE1 overexpression stimulates MMP3 secretion but does not change transmigration through a fixed substrate, MV3 cell invasion of a native substrate depends on MMP activity rather than on a modifiable cortical stiffness.

Pharmacological reversal of renal cysts from secretion to absorption suggests a potential therapeutic strategy for managing autosomal dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) induces a secretory phenotype, resulting in multiple fluid-filled cysts. We have previously demonstrated that VX-809, a corrector of the cystic fibrosis transmembrane conductance regulator (CFTR), reduces cyst growth. Here, we show that in normal mice CFTR is located within the cells and also at the apical and basolateral membranes. However, in polycystic kidney disease (pkd1)-knockout mice, CFTR was located at the plasma membrane, consistent with its role in cAMP-dependent fluid secretion. In cystic mice, VX-809 treatment increased CFTR levels at the apical membrane and reduced its association with the endoplasmic reticulum. Surprisingly, VX-809 treatment significantly increased CFTR's co-localization with the basolateral membrane in cystic mice. Na+/H+ exchanger 3 (NHE3) is present in pkd1-knockout and normal mice and in proximal tubule-derived, cultured pkd1-knockout cells. VX-809 increased the expression, activity, and apical plasma membrane localization of NHE3. Co-localization of epithelial sodium channel (ENaC) with the plasma membrane was reduced in cysts in pkd1-knockout mice, consistent with an inability of the cysts to absorb fluid. Interestingly, in the cystic mice, VX-809 treatment increased ENaC levels at the apical plasma membrane consistent with fluid absorption. Thus, VX-809 treatment of pkd1-null mouse kidneys significantly affected CFTR, NHE3, and ENaC, altering the cyst phenotype from one poised toward fluid secretion toward one more favorable for absorption. VX-809 also altered the location of CFTR but not of NHE3 or ENaC in normal mice. Given that VX-809 administration is safe, it may have potential utility for treating patients with ADPKD.

MicroRNA-19a attenuates hypoxia-induced cardiomyocyte apoptosis by downregulating NHE-1 expression and decreasing calcium overload.

miR-19a has been shown to be involved in coronary microvascular obstruction injury; however, the underlying molecular mechanisms remain unknown. In our study, we tried to explore the role of miR-19a in cardiomyocyte apoptosis and calcium overload in vivo and in vitro induced by hypoxia. We established the acute myocardial infarction (AMI) rat model by ligating the left anterior descending artery. The expression of miR-19a in the infarct zone of AMI rats and myocardial tissue in the same position in sham rats was analyzed using RT-qPCR while Na(+) /H(+) exchanger 1 (NHE-1) was detected by Western blotting. We also observed the effects of overexpressing miR-19a or administering an NHE-1 inhibitor (cariporide) on hypoxia-induced (HI) calcium overload and apoptosis in primary cardiomyocytes. In addition, dual-luciferase reporter assays were conducted to investigate the potential target of miR-19a on NHE-1. Decreased miR-19a expression, as well as increased apoptosis and NHE-1 expression, were observed in the AMI model. Furthermore, after hypoxia stimulation, miR-19a was gradually reduced as time increased in primary cardiomyocytes. Overexpressing miR-19a using mimics ameliorated the increase in NHE-1 in hypoxic cardiomyocytes and thereby reduced the HI cell calcium overload and cell apoptosis rate from 12.32% to 9.5% (P < .01). In addition, the dual-luciferase reporter gene assay results verified that NHE-1 was the direct target of miR-19a.Our findings suggest that miR-19a activation can attenuate HI cardiomyocyte apoptosis by downregulating NHE-1 expression and decreasing calcium overload.

A20 in Myeloid Cells Protects Against Hypertension by Inhibiting Dendritic Cell-Mediated T-Cell Activation.

The ubiquitin-editing protein A20 in dendritic cells (DCs) suppresses NF-κB (nuclear factor-κB) signaling and constrains DC-mediated T-cell stimulation, but the role of A20 in modulating the hypertensive response requires elucidation. Here, we tested the hypothesis that A20 in CD11c-expressing myeloid cells mitigates Ang II (angiotensin II)-induced hypertension by limiting renal T-cell activation. Mice with heterozygous deletion of A20 in CD11c-expressing myeloid cells (DC ACT[Cd11c-Cre+A20flox/wt]) have spontaneous DC activation but have normal baseline blood pressures. In response to low-dose chronic Ang II infusion, DC ACT mice compared with WT (wild type) controls had an exaggerated hypertensive response and augmented proportions of CD62LloCD44hi effector memory T lymphocytes in the kidney lymph node. After 10 days of Ang II, DC ACT kidneys had increased numbers of memory effector CD8+, but not CD4+ T cells, compared with WTs. Moreover, the expressions of TNF-α (tumor necrosis factor-α) and IFN-γ (interferon-γ) were upregulated in the DC ACT renal CD8+ T cells but not CD4+ T cells. Saline challenge testing revealed enhanced renal fluid retention in the DC ACT mice. DC ACT kidneys showed augmented protein expression of γ-epithelial sodium channel and NHE3 (sodium-hydrogen antiporter 3). DC ACT mice also had greater reductions in renal blood flow following acute injections with Ang II and enhanced oxidant stress in the vasculature as evidenced by higher circulating levels of malondialdehyde compared with WT controls. To directly test whether enhanced T-cell activation in the DC ACT cohort was responsible for their exaggerated hypertensive response, we chronically infused Ang II into lymphocyte-deficient DC ACT Rag1 (recombination activating protein 1)-deficient (Rag1-/-) mice and WT (Cd11c-Cre-A20flox/wt) Rag1-/- controls. The difference in blood pressure elevation accruing from DC activation was abrogated on the Rag1-/- strain. Following stimulation of the renin-angiotensin system, A20 suppresses DC activation and thereby mitigates T-cell-dependent blood pressure elevation.