Acidic preconditioning induced intracellular acid adaptation to protect renal injury via dynamic phosphorylation of focal adhesion kinase-dependent activation of sodium hydrogen exchanger 1.

BACKGROUND: Disruptions in intracellular pH (pHi) homeostasis, causing deviations from the physiological range, can damage renal epithelial cells. However, the existence of an adaptive mechanism to restore pHi to normalcy remains unclear. Early research identified H+ as a critical mediator of ischemic preconditioning (IPC), leading to the concept of acidic preconditioning (AP). This concept proposes that short-term, repetitive acidic stimulation can enhance a cell's capacity to withstand subsequent adverse stress. While AP has demonstrated protective effects in various ischemia-reperfusion (I/R) injury models, its application in kidney injury remains largely unexplored. METHODS: An AP model was established in human kidney (HK2) cells by treating them with an acidic medium for 12 h, followed by a recovery period with a normal medium for 6 h. To induce hypoxia/reoxygenation (H/R) injury, HK2 cells were subjected to hypoxia for 24 h and reoxygenation for 1 h. In vivo, a mouse model of IPC was established by clamping the bilateral renal pedicles for 15 min, followed by reperfusion for 4 days. Conversely, the I/R model involved clamping the bilateral renal pedicles for 35 min and reperfusion for 24 h. Western blotting was employed to evaluate the expression levels of cleaved caspase 3, cleaved caspase 9, NHE1, KIM1, FAK, and NOX4. A pH-sensitive fluorescent probe was used to measure pHi, while a Hemin/CNF microelectrode monitored kidney tissue pH. Immunofluorescence staining was performed to visualize the localization of NHE1, NOX4, and FAK, along with the actin cytoskeleton structure in HK2 cells. Cell adhesion and scratch assays were conducted to assess cell motility. RESULTS: Our findings demonstrated that AP could effectively mitigate H/R injury in HK2 cells. This protective effect and the maintenance of pHi homeostasis by AP involved the upregulation of Na+/H+ exchanger 1 (NHE1) expression and activity. The activity of NHE1 was regulated by dynamic changes in pHi-dependent phosphorylation of Focal Adhesion Kinase (FAK) at Y397. This process was associated with NOX4-mediated reactive oxygen species (ROS) production. Furthermore, AP induced the co-localization of FAK, NOX4, and NHE1 in focal adhesions, promoting cytoskeletal remodeling and enhancing cell adhesion and migration capabilities. CONCLUSIONS: This study provides compelling evidence that AP maintains pHi homeostasis and promotes cytoskeletal remodeling through FAK/NOX4/NHE1 signaling. This signaling pathway ultimately contributes to alleviated H/R injury in HK2 cells.

NHE1 regulation in NAFLD in vitro contributes to hepatocyte injury and HSC crosstalk.

Non-alcoholic fatty liver disease (NAFLD) is the fastest-growing cause of liver-associated death globally. Whole-body knockout (KO) of Na+/H+ exchanger 1 (NHE1, SLC9A1) was previously proposed to protect against high-fat diet-induced liver damage; however, mechanistic insight was lacking. The aim of the present work was to address this question in vitro to determine how NHE1, specifically in hepatocytes, impacts lipid overload-induced inflammation, fibrosis, and hepatocyte-hepatic stellate cell (HSC) crosstalk. We induced palmitate (PA)-based steatosis in AML12 and HepG2 hepatocytes; manipulated NHE1 activity pharmacologically and by CRISPR/Cas9-mediated KO and overexpression; and measured intracellular pH (pHi), steatosis-associated inflammatory and fibrotic mediators, and cell death. PA treatment increased NHE1 mRNA levels but modestly reduced NHE1 protein expression and hepatocyte pHi. NHE1 KO in hepatocytes did not alter lipid droplet accumulation but reduced inflammatory signaling (p38 MAPK activity), lipotoxicity (4-HNE accumulation), and apoptosis (poly-ADP-ribose-polymerase-1 (PARP) cleavage). Conditioned medium from PA-treated hepatocytes increased the expression of NHE1 and of the fibrosis regulator tissue inhibitor of matrix metalloproteinases-2 in LX-2 HSCs, in a manner abolished by NHE1 KO in hepatocytes. We conclude that NHE1 is regulated in NAFLD in vitro and contributes to the ensuing damage by aggravating hepatocyte injury and stimulating hepatocyte-HSC crosstalk.

Na+/H+-exchange inhibition by cariporide is compensated via Na+,HCO3--cotransport and has no net growth consequences for ErbB2-driven breast carcinomas.

Defense against intracellular acidification of breast cancer tissue depends on net acid extrusion via Na+,HCO3--cotransporter NBCn1/Slc4a7 and Na+/H+-exchanger NHE1/Slc9a1. NBCn1 is increasingly recognized as breast cancer susceptibility protein and promising therapeutic target, whereas evidence for targeting NHE1 is discordant. Currently, selective small molecule inhibitors exist against NHE1 but not NBCn1. Cellular assays-with some discrepancies-link NHE1 activity to proliferation, migration, and invasion; and disrupted NHE1 expression can reduce triple-negative breast cancer growth. Studies on human breast cancer tissue associate high NHE1 expression with reduced metastasis and-in some molecular subtypes-improved patient survival. Here, we evaluate Na+/H+-exchange and therapeutic potential of the NHE1 inhibitor cariporide/HOE-642 in murine ErbB2-driven breast cancer. Ex vivo, cariporide inhibits net acid extrusion in breast cancer tissue (IC50 = 0.18 µM) and causes small decreases in steady-state intracellular pH (pHi). In vivo, we deliver cariporide orally, by osmotic minipumps, and by intra- and peritumoral injections to address the low oral bioavailability and fast metabolism. Prolonged cariporide administration in vivo upregulates NBCn1 expression, shifts pHi regulation towards CO2/HCO3--dependent mechanisms, and shows no net effect on the growth rate of ErbB2-driven primary breast carcinomas. Cariporide also does not influence proliferation markers in breast cancer tissue. Oral, but not parenteral, cariporide elevates serum glucose by ∼1.5 mM. In conclusion, acute administration of cariporide ex vivo powerfully inhibits net acid extrusion from breast cancer tissue but lowers steady-state pHi minimally. Prolonged cariporide administration in vivo is compensated via NBCn1 and we observe no discernible effect on growth of ErbB2-driven breast carcinomas.

Neutrophils actively swell to potentiate rapid migration.

While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.

Empagliflozin prevents heart failure through inhibition of the NHE1-NO pathway, independent of SGLT2.

Sodium glucose cotransporter 2 inhibitors (SGLT2i) constitute the only medication class that consistently prevents or attenuates human heart failure (HF) independent of ejection fraction. We have suggested earlier that the protective mechanisms of the SGLT2i Empagliflozin (EMPA) are mediated through reductions in the sodium hydrogen exchanger 1 (NHE1)-nitric oxide (NO) pathway, independent of SGLT2. Here, we examined the role of SGLT2, NHE1 and NO in a murine TAC/DOCA model of HF. SGLT2 knockout mice only showed attenuated systolic dysfunction without having an effect on other signs of HF. EMPA protected against systolic and diastolic dysfunction, hypertrophy, fibrosis, increased Nppa/Nppb mRNA expression and lung/liver edema. In addition, EMPA prevented increases in oxidative stress, sodium calcium exchanger expression and calcium/calmodulin-dependent protein kinase II activation to an equal degree in WT and SGLT2 KO animals. In particular, while NHE1 activity was increased in isolated cardiomyocytes from untreated HF, EMPA treatment prevented this. Since SGLT2 is not required for the protective effects of EMPA, the pathway between NHE1 and NO was further explored in SGLT2 KO animals. In vivo treatment with the specific NHE1-inhibitor Cariporide mimicked the protection by EMPA, without additional protection by EMPA. On the other hand, in vivo inhibition of NOS with L-NAME deteriorated HF and prevented protection by EMPA. In conclusion, the data support that the beneficial effects of EMPA are mediated through the NHE1-NO pathway in TAC/DOCA-induced heart failure and not through SGLT2 inhibition.

Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma.

OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

Employing the sustained-release properties of poly(lactic-co-glycolic acid) nanoparticles to reveal a novel mechanism of sodium-hydrogen exchanger 1 in neuropathic pain.

Neuropathic pain is caused by injury or disease of the somatosensory system, and its course is usually chronic. Several studies have been dedicated to investigating neuropathic pain-related targets; however, little attention has been paid to the persistent alterations that these targets, some of which may be crucial to the pathophysiology of neuropathic pain. The present study aimed to identify potential targets that may play a crucial role in neuropathic pain and validate their long-term impact. Through bioinformatics analysis of RNA sequencing results, we identified Slc9a1 and validated the reduced expression of sodium-hydrogen exchanger 1 (NHE1), the protein that Slc9a1 encodes, in the spinal nerve ligation (SNL) model. Colocalization analysis revealed that NHE1 is primarily co-localized with vesicular glutamate transporter 2-positive neurons. In vitro experiments confirmed that poly(lactic-co-glycolic acid) nanoparticles loaded with siRNA successfully inhibited NHE1 in SH-SY5Y cells, lowered intracellular pH, and increased intracellular calcium concentrations. In vivo experiments showed that sustained suppression of spinal NHE1 expression by siRNA-loaded nanoparticles resulted in delayed hyperalgesia in naïve and SNL model rats, whereas amiloride-induced transient suppression of NHE1 expression yielded no significant changes in pain sensitivity. We identified Slc9a1, which encodes NHE1, as a key gene in neuropathic pain. Utilizing the sustained release properties of nanoparticles enabled us to elucidate the chronic role of decreased NHE1 expression, establishing its significance in the mechanisms of neuropathic pain.

Targeting Na-H exchanger 1 overcomes nuclear factor kappa B-mediated tumor resistance to radiotherapy.

Intrinsic or acquired radioresistance often limits the efficacy of radiation therapy (RT), thereby leading to local control failure. Cancerous cells have abnormal pH dynamics due to high metabolic demands, but it is unclear how pH dynamics contribute to radioresistance. In this study, we investigated the role of Na-H exchange 1 (NHE1), the major intracellular pH (pHi) regulator, in RT response. We observed that RT increased NHE1 expression and modulated pHi in MDA-MB-231 human breast cancer cells. When combined with RT, pharmacological NHE1 inhibition by 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) reduced pHi and clonogenic survival. EIPA attenuated radiation-damaged DNA repair, increasing G2/M cell cycle arrest. The combination of EIPA and RT increased apoptotic cell death while decreasing phosphorylation of NF-κB p65. Similarly, the knockdown of NHE1 increased radiosensitivity with lower pHi and increased apoptosis. Consistent with in vitro data, the EIPA plus RT inhibited the growth of MDA-MB-231 xenograft tumors in mice to a greater extent than either EIPA or RT alone. EIPA abrogated the RT-induced increase in NHE1 and phospho-NF-κB p65 expression in tumor tissues. Such coincidence of increased NHE1 level, pHi, and NF-κB activation was also found in radioresistant MDA-MB-231 cells, which were reversed by EIPA treatment. Bioinformatics analysis of RNA sequencing data revealed that inhibiting NHE1 reversed three core gene networks that were up-regulated in radioresistant cells and correlated with high NHE1 expression in patient samples: NF-κB, senescence, and extracellular matrix. Taken together, our findings suggest that NHE1 contributes to RT resistance via NF-κB-mediated signaling networks, and NHE1 may be a promising target for improving RT outcomes.

Pulmonary endothelial cells from different vascular segments exhibit unique recovery from acidification and Na+/H+ exchanger isoform expression.

Sodium-hydrogen exchangers (NHEs) tightly regulate intracellular pH (pHi), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined. Utilizing the ammonium-prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited unique recovery rates from the acid load dependent upon the concentration of the sodium transport inhibitor, amiloride; further, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium-dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two-gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular versus pulmonary artery endothelial cells, which was confirmed at the protein level. Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.

Permissive role of Na+/H+ exchanger isoform 1 in migration and invasion of triple-negative basal-like breast cancer cells.

In breast cancer, it is the resulting metastasis that is the primary cause of fatality. pH regulatory proteins and the tumor microenvironment play an important role in metastasis of cancer cells and acid-extruding proteins are critical in this process. There are several types of breast cancer and triple-negative breast cancer tends to be more metastatic and invasive and is itself is composed of several types. MDA-MB-468 are a triple-negative breast cancer cell line and are classified as basal-like and basal tumors account for up to 15% of breast cancers. Here we examined the effect of removal of the acid-extruding protein, the Na+/H+ exchanger isoform one, from MDA-MB-468 cells. NHE1 was deleted from these cells using the CRISPR/Cas9 system. Western blotting and measurement of activity confirmed the absence of the protein. In wounding/cell migration experiments, deletion of NHE1 reduced the rate of cell migration in the presence of low- or high-serum concentrations. Anchorage-dependent colony formation was also greatly reduced by deletion of the NHE1 protein. Cell proliferation was not affected by knockout of NHE1. The results demonstrate that NHE1 has an important role in migration and invasion of basal-like triple-negative breast cancer cells.

Hypoxia- and hyperoxia-related gene expression dynamics during developmental critical windows of the tropical gar Atractosteus tropicus.

Aquatic hypoxia is both a naturally-occurring and anthropogenically-generated event. Fish species have evolved different adaptations to cope with hypoxic environments, including gill modifications and air breathing. However, little is known about the molecular mechanisms involved in the respiration of embryonic and larval fishes during critical windows of development. We assessed expression of the genes hif-1α, fih-1, nhe1, epo, gr and il8 using the developing tropical gar as a piscine model during three developmental periods (fertilization to hatch, 1 to 6 days post hatch (dph) and 7 to 12 dph) when exposed to normoxia (~7.43 mg/L DO), hypoxia (~2.5 mg/L DO) or hyperoxia (~9.15 mg/L DO). All genes had higher expression when fish were exposed to either hypoxia or hyperoxia during the first two developmental periods. However, fish continuously exposed to hypoxia had increased expression of the six genes by hatching and 6 dph, and by 12 dph only hif-1α still had increased expression. The middle developmental period was the most hypoxia-sensitive, coinciding with several changes in physiology and morphology. The oldest larvae were the most resilient to gene expression change, with little variation in expression of the six genes compared. This study is the first to relate the molecular response of an air-breathing fish to oxygen availability to developmental critical windows and contributes to our understanding of some molecular responses of developing fish to changes in oxygen availability.

Roles of the Na+/H+ Exchanger Isoform 1 and Urokinase in Prostate Cancer Cell Migration and Invasion.

Prostate cancer is a leading cause of cancer-associated deaths in men over 60 years of age. Most patients are killed by tumor metastasis. Recent evidence has implicated a role of the tumor microenvironment and urokinase plasminogen activator (uPA) in cancer cell migration, invasion, and metastasis. Here, we examine the role of the Na+/H+ exchanger isoform 1 (NHE1) and uPA in DU 145 prostate cancer cell migration and colony formation. Knockout of NHE1 reduced cell migration. The effects of a series of novel NHE1/uPA hexamethylene-amiloride-based inhibitors with varying efficacy towards NHE1 and uPA were examined on prostate cancer cells. Inhibition of NHE1-alone, or with inhibitors combining NHE1 or uPA inhibition-generally did not prevent prostate cancer cell migration. However, uPA inhibition-but not NHE1 inhibition-prevented anchorage-dependent colony formation. Application of inhibitors at concentrations that only saturate uPA inhibition decreased tumor invasion in vivo. The results suggest that while knockout of NHE1 affects cell migration, these effects are not due to NHE1-dependent proton translocation. Additionally, while neither NHE1 nor uPA activity was critical in cell migration, only uPA activity appeared to be critical in anchorage-dependent colony formation of DU 145 prostate cancer cells and invasion in vivo.

Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783-790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.

Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett's Esophageal Cell Line.

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett's esophagus, and this is presumably a compensatory mechanism against this toxic agent.

Contributions of Sodium-Hydrogen Exchanger 1 and Mitogen-Activated Protein Kinases to Enhanced Retinal Venular Constriction to Endothelin-1 in Diabetes.

Diabetes elevates endothelin-1 (ET-1) in the vitreous and enhances constriction of retinal venules to this peptide. However, mechanisms contributing to ET-1-induced constriction of retinal venules are incompletely understood. We examined roles of sodium-hydrogen exchanger 1 (NHE1), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and extracellular calcium (Ca2+) in retinal venular constriction to ET-1 and the impact of diabetes on these signaling molecules. Retinal venules were isolated from control pigs and pigs with streptozocin-induced diabetes for in vitro studies. ET-1-induced vasoconstriction was abolished in the absence of extracellular Ca2+ and sensitive to c-Jun N-terminal kinase (JNK) inhibitor SP600125 but unaffected by extracellular signal-regulated kinase (ERK) inhibitor PD98059, p38 kinase inhibitor SB203580, or broad-spectrum PKC inhibitor Gö 6983. Diabetes (after 2 weeks) enhanced venular constriction to ET-1, which was insensitive to PD98059 and Gö 6983 but was prevented by NHE1 inhibitor cariporide, SB203580, and SP600125. In conclusion, extracellular Ca2+ entry and activation of JNK, independent of ERK and PKC, mediate constriction of retinal venules to ET-1. Diabetes activates p38 MAPK and NHE1, which cause enhanced venular constriction to ET-1. Treatments targeting these vascular molecules may lessen retinal complications in early diabetes.

Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival.

Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3- cotransport and Na+/H+ exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+ exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3- cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+ exchanger NHE1/SLC9A1 and Na+,HCO3- cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3- cotransport associates with severe breast cancer: enlarged CO2/HCO3--dependent rises in pHi predict accelerated cell proliferation, whereas enhanced CO2/HCO3--dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from luminal A or basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3- cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.

Sodium Glucose Co-Transporter 2 Inhibitors Ameliorate Endothelium Barrier Dysfunction Induced by Cyclic Stretch through Inhibition of Reactive Oxygen Species.

SGLT-2i's exert direct anti-inflammatory and anti-oxidative effects on resting endothelial cells. However, endothelial cells are constantly exposed to mechanical forces such as cyclic stretch. Enhanced stretch increases the production of reactive oxygen species (ROS) and thereby impairs endothelial barrier function. We hypothesized that the SGLT-2i's empagliflozin (EMPA), dapagliflozin (DAPA) and canagliflozin (CANA) exert an anti-oxidative effect and alleviate cyclic stretch-induced endothelial permeability in human coronary artery endothelial cells (HCAECs). HCAECs were pre-incubated with one of the SGLT-2i's (1 µM EMPA, 1 µM DAPA and 3 µM CANA) for 2 h, followed by 10% stretch for 24 h. HCAECs exposed to 5% stretch were considered as control. Involvement of ROS was measured using N-acetyl-l-cysteine (NAC). The sodium-hydrogen exchanger 1 (NHE1) and NADPH oxidases (NOXs) were inhibited by cariporide, or GKT136901, respectively. Cell permeability and ROS were investigated by fluorescence intensity imaging. Cell permeability and ROS production were increased by 10% stretch; EMPA, DAPA and CANA decreased this effect significantly. Cariporide and GKT136901 inhibited stretch-induced ROS production but neither of them further reduced ROS production when combined with EMPA. SGLT-2i's improve the barrier dysfunction of HCAECs under enhanced stretch and this effect might be mediated through scavenging of ROS. Anti-oxidative effect of SGLT-2i's might be partially mediated by inhibition of NHE1 and NOXs.

Type-1 Na+/H+ exchanger is a prognostic factor and associate with immune infiltration in liver hepatocellular carcinoma.

AIMS: SLC9A1 plays an important role in the growth, differentiation and glycolysis of tumor cells. The present study aimed to elucidate the correlation between SLC9A1 and tumor immune infiltration. MAIN METHODS: Expression level of SLC9A1 gene in tumors was identified in GEPIA. The correlation between SLC9A1 and survival in various types of cancers was analyzed by the PrognoScan. SLC9A1 immune infiltration levels and clinical correlation analysis was generated via TIMER and TIMER2.0. KEGG enrichment analysis of SLC9A1 expression was evaluated via STRING. KEY FINDINGS: We found that, in cancers such as liver hepatocellular carcinoma (LIHC), the expression of SLC9A1 was significantly higher in tumor tissues compared with normal tissues, and was significantly associated with poor prognosis. Further analysis showed that SLC9A1 expression in LIHC was significantly positively correlated with immune cell infiltration, and the correlation was the highest for LIHC among 40 cancers. The expression of SLC9A1 is significantly correlated with the immune marker set of most immune cells in LIHC. Furthermore, we found that the expression level of TGF-ß (TGFB1) in Tregs showed the highest correlation with the expression of SLC9A1 in LIHC. SIGNIFICANCE: The increased expression of SLC9A1 is positively correlated with the prognosis of cancer and the level of immune infiltration. Therefore, SLC9A1 is an important prognostic factor for immunotherapy against hepatocellular carcinoma.

Ethyl isopropyl amiloride decreases oxidative phosphorylation and increases mitochondrial fusion in clonal untransformed and cancer cells.

Many cancer cells, regardless of their tissue origin or genetic landscape, have increased expression or activity of the plasma membrane Na-H exchanger NHE1 and a higher intracellular pH (pHi) compared with untransformed cells. A current perspective that remains to be validated is that increased NHE1 activity and pHi enable a Warburg-like metabolic reprogramming of increased glycolysis and decreased mitochondrial oxidative phosphorylation. We tested this perspective and find it is not accurate for clonal pancreatic and breast cancer cells. Using the pharmacological reagent ethyl isopropyl amiloride (EIPA) to inhibit NHE1 activity and decrease pHi, we observe no change in glycolysis, as indicated by secreted lactate and intracellular pyruvate, despite confirming increased activity of the glycolytic enzyme phosphofructokinase-1 at higher pH. Also, in contrast to predictions, we find a significant decrease in oxidative phosphorylation with EIPA, as indicated by oxygen consumption rate (OCR). Decreased OCR with EIPA is not associated with changes in pathways that fuel oxidative phosphorylation or with mitochondrial membrane potential but occurs with a change in mitochondrial dynamics that includes a significant increase in elongated mitochondrial networks, suggesting increased fusion. These findings conflict with current paradigms on increased pHi inhibiting oxidative phosphorylation and increased oxidative phosphorylation being associated with mitochondrial fusion. Moreover, these findings raise questions on the suggested use of EIPA-like compounds to limit metabolic reprogramming in cancer cells.

Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective Inhibitors.

The K+-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane pH that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine 24 uPA IC50 = 175 nM, NHE1 IC50 = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine 26 uPA IC50 = 86 nM, NHE1 IC50 = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino (29 NHE1 IC50 = 129 nM, uPA IC50 = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) (30 NHE1 IC50 = 85 nM, uPA IC50 = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.