NHE3 Protein Research Articles

Cardiac sodium/hydrogen exchanger (NHE11) as a novel potential target for SGLT2i in heart failure

Abstract Background Despite the cardiac benefits of sodium/glucose cotransporter 2 inhibitors (SGLT2i), their basic of action remains unclear. Sodium/hydrogen exchanger (NHE) has been proposed as mechanism of action of SGLT2i, but there are controversies related to its function and expression in heart failure (HF). Purpose We hypothesized that sodium transported-related molecules could be altered in human HF and they could be modulated through SGLT2i. Methods Transcriptome-level differences in genes involved in sodium transport between HF and control (CNT) were investigated in 36 heart samples with RNA-sequencing technology (HF, n=26; CNT, n=10). In addition, NHE11 and NHE1 protein levels were determined in 80 heart samples (HF, n=70; CNT, n=10) by ELISA assay. Furthermore, the effect of empagliflozin on NHE11 mRNA levels in rat left ventricular tissue (n=22) was studied through RT-qPCR. Results We observed alterations in several genes involved in sodium transport. Among them the overexpression in SLC9C2 (p=0.005) and SCL9A1 (p=0.020) genes, which encode the NHE11 and NHE1 proteins, respectively. In addition, cardiac protein levels of these molecules were determined. We found a significant increase in the concentration of NHE11 (p=0.042) and NHE1 (p=0.018) in HF. Moreover, NHE11 levels were correlated with left ventricular diameters. Due to the relevance of NHE11 changes observed, we studied the effect of SGLTi on its expression. NHE11 mRNA levels were reduced in rats treated with empagliflozin (p=0.010). Conclusions Our findings show alterations in several sodium transport, reinforce the importance of these channels in HF progression. We described upregulation in NHE11 and NHE1 in HF patients, but only NHE11 correlated with cardiac dysfunction. In addition, the most relevant finding is the change observed in the expression of the unknown NHE11 after treatment with empagliflozin. These results propose NHE11 as a potential target of SGLT2i in cardiac tissue. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Downregulation of NHE-3 (SLC9A3) expression by MicroRNAs in intestinal epithelial cells.

Na+/H+ exchanger-3 (NHE-3) is the major apical membrane transporter involved in vectorial Na+ absorption in the intestine. Dysregulation of NHE-3 expression and/or function has been implicated in pathophysiology of diarrhea associated with gut inflammation and infections. Therefore, it is critical to understand the mechanisms involved in the regulation of NHE-3 expression. MicroRNAs (miRNAs) are highly conserved small RNAs that can regulate gene expression at the posttranscriptional level. To date, however, very little is known about the regulation of NHE-3 expression by microRNAs. Therefore, current studies were undertaken to examine the potential miRNA candidates that can regulate the expression of NHE-3 in intestinal epithelial cells. In silico analysis, using different algorithms, predicted several miRNAs that target NHE-3. MicroRNAs with highest context and target score, miR-326, miR-744-5p, and miR-330-5p, were selected for the current study. Human NHE-3 gene 3' untranslated region [3'UTR; 160 base pair (bp)] was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with mimics of miR-326, miR-744-5p, and miR-330-5p into Caco-2, HT-29, and SK-CO15 cells. Cotransfection of NHE-3 3' UTR with miR-326 and -miR-330-5p mimics resulted in a significant decrease in relative luciferase activity. Transfection of miR-326 and -330-5p mimics into SK-CO15 cells significantly decreased the NHE-3 protein expression, with no change in NHE-3 messenger ribonucleic acid (mRNA) levels. Our findings demonstrate a novel mechanism for posttranscriptional regulation of NHE-3 by miR-326 and -330-5p by translational repression. We speculate that miR-326 and -330-5p dependent pathways may be involved in modulating NHE-3 expression under physiological and pathophysiological conditions.

Cardiac Sodium/Hydrogen Exchanger (NHE11) as a Novel Potential Target for SGLT2i in Heart Failure: A Preliminary Study.

Despite the reduction of cardiovascular events, including the risk of death, associated with sodium/glucose cotransporter 2 inhibitors (SGLT2i), their basic action remains unclear. Sodium/hydrogen exchanger (NHE) has been proposed as the mechanism of action, but there are controversies related to its function and expression in heart failure (HF). We hypothesized that sodium transported-related molecules could be altered in HF and modulated through SGLT2i. Transcriptome alterations in genes involved in sodium transport in HF were investigated in human heart samples by RNA-sequencing. NHE11 and NHE1 protein levels were determined by ELISA; the effect of empagliflozin on NHE11 and NHE1 mRNA levels in rats’ left ventricular tissues was studied through RT-qPCR. We highlighted the overexpression of SLC9C2 and SCL9A1 sodium transport genes and the increase of the proteins that encode them (NHE11 and NHE1). NHE11 levels were correlated with left ventricular diameters, so we studied the effect of SGLT2i on its expression, observing that NHE11 mRNA levels were reduced in treated rats. We showed alterations in several sodium transports and reinforced the importance of these channels in HF progression. We described upregulation in NHE11 and NHE1, but only NHE11 correlated with human cardiac dysfunction, and its levels were reduced after treatment with empagliflozin. These results propose NHE11 as a potential target of SGLT2i in cardiac tissue.

The Remaining Conundrum of the Role of the Na+/H+ Exchanger Isoform 1 (NHE1) in Cardiac Physiology and Pathology: Can It Be Rectified?

The mammalian exchanger (NHE) is a family of ubiquitous membrane proteins present in humans. Isoform one (NHE1) is present on the plasma membrane and regulates intracellular pH by removal of one intracellular proton in exchange for one extracellular sodium thus functioning as an electroneutral process. Human NHE1 has a 500 amino acid membrane domain plus a C-terminal 315 amino acid, regulatory cytosolic tail. It is regulated through a cytosolic regulatory C-terminal tail which is subject to phosphorylation and is modulated by proteins and lipids. Substantial evidence has implicated NHE1 activity in both myocardial ischemia and reperfusion damage and myocardial remodeling resulting in heart failure. Experimental data show excellent cardioprotection with NHE1 inhibitors although results from clinical results have been mixed. In cardiac surgery patients receiving the NHE1 inhibitor cariporide, subgroups showed beneficial effects of treatment. However, in one trial this was associated with a significantly increased incidence of ischemic strokes. This likely reflected both inappropriate dosing regimens as well as overly high drug doses. We suggest that further progress towards NHE1 inhibition as a treatment for cardiovascular disease is warranted through the development of novel compounds to inhibit NHE1 that are structurally different than those previously used in compromised clinical trials. Some novel pyrazinoyl guanidine inhibitors of NHE1 are already in development and the recent elucidation of the three-dimensional structure of the NHE1 protein and identity of the inhibitor binding site may facilitate development. An alternative approach may also be to control the endogenous regulation of activity of NHE1, which is activated in disease.

Structural basis of autoinhibition of the human NHE3-CHP1 complex.

Sodium-proton exchanger 3 (NHE3/SLC9A3) located in the apical membrane of renal and gastrointestinal epithelia mediates salt and fluid absorption and regulates pH homeostasis. As an auxiliary regulatory factor of NHE proteins, calcineurin B homologous protein 1 (CHP1) facilitates NHE3 maturation, plasmalemmal expression, and pH sensitivity. Dysfunctions of NHE3 are associated with renal and digestive system disorders. Here, we report the cryo–electron microscopy structure of the human NHE3-CHP1 complex in its inward-facing conformation. We found that a cytosolic helix-loop-helix motif in NHE3 blocks the intracellular cavity formed between the core and dimerization domains, functioning as an autoinhibitory element and hindering substrate transport. Furthermore, two phosphatidylinositol molecules are found to bind to the peripheric juxtamembrane sides of the complex, function as anchors to stabilize the complex, and may thus enhance its transport activity.

Phylogenetic and protein prediction analysis reveals the taxonomically diverse distribution of virulence factors in Bacillus cereus strains.

Bacillus cereus is a food contaminant with widely varying enterotoxic potential due to its virulence proteins. In this article, phylogenetic analysis of the amino acid sequences from the whole-genomes of 41 strains, evolutionary distance calculation of the amino acid sequences of the virulence genes, and functional and structural predictions of the virulence proteins were performed to reveal the taxonomically diverse distribution of virulence factors. The genome evolution of the strains showed a clustering trend based on the protein-coding virulence genes. The strains of B. cereus have evolved into non-toxic risk and toxic risk clusters with medium-high- and medium-low-risk subclusters. The evolutionary transfer distances of incomplete virulence genes relative to housekeeping genes were greater than those of complete virulence genes, and the distance values of HblACD were higher than those of nheABC and CytK among the complete virulence genes. Cytoplasmic localization was impossible for all the virulence proteins, and NheB, NheC, Hbl-B, and Hbl-L1 were predicted to be extracellular. Nhe and Hbl proteins except CytK had similar spatial structures. The predicted structures of Nhe and Hbl mainly showed ‘head’ and ‘tail’ domains. The ‘head’ of NheA and Hbl-B, including two α-helices separated by β-tongue strands, might play a special role in the formation of Nhe trimers and Hbl trimers, respectively. The ‘cap’ of CytK, which includes two ‘latches’ with many β-sheets, formed a β-barrel structure with pores, and a ‘rim’ balanced the structure. The evolution of B. cereus strains showed a clustering tendency based on the protein-coding virulence genes, and the complete virulence-gene operon combination had higher relative genetic stability. The beta-tongue or latch associated with β-sheet folding might play an important role in the binding of virulence structures and pore-forming toxins in B. cereus.

Inhibition of Na‐H exchanger 3 ameliorates lower limb ischemia/reperfusion‐induced acute kidney injury through preservation of mitochondrial biogenesis in mice

Despite advances in our understanding of acute kidney injury (AKI), its mortality rate remains high. Novel therapeutic targets are therefore needed. Mitochondrial dysfunction is a pivotal factor in the pathogenesis of AKI. The Na+‐H+ exchanger 3 (NHE3) plays a critical role in ATP‐dependent Na+ absorption in the proximal tubules. As such, inhibition of NHE3 may reduce metabolic demand for mitochondria thus reducing mitochondrial stress and dysfunction. In the present study, we sought to determine whether inhibition of NHE3 alleviated lower limb ischemia/reperfusion‐induced AKI. We induced lower limb ischemia/reperfusion by applying tourniquets bilaterally on the inguinal areas of male B6 or transgenic mice for 76 minutes followed by monitoring in metabolic cages for 22 hours. Control mice were treated in the same way except for no tourniquets. We used acetazolamide and kidney epithelial cell‐specific knockout to inhibit NHE3 in mice and monensin to mimic NHE3 in LLC‐PK1 cells. We assessed the renal function with urine output, transcutaneous GFR (tGFR) and serum urea nitrogen (SUN) and histology with H&E staining. We measured mitochondrial biogenesis by mRNA levels of PGC‐1α, a master regulator of mitochondrial biogenesis, and its targeted genes NDUFS1 and ATP5o. Compared with the control, tourniquets reduced urinary output by 62%, tGFR by 95% and increased SUN by 774% in B6 mice. Acetazolamide, given by oral gavage (40 mg/kg), reduced NHE3 protein abundance by 37% in the kidney cortex of tourniquet‐injured mice, attenuated tourniquet‐induced reductions in urinary output by 100% and tGFR by 77%, and increase in SUN by 76%. It also attenuated tourniquet‐induced peritubular focal hemorrhage, tubular degeneration and nuclear condensation in the renal cortex. Tourniquets reduced mRNA levels of PGC‐1α, NDUFS1 and ATP5o by 45%, 44%, and 52%, respectively. Acetazolamide significantly blunted all these effects. Compared with no tourniquet‐injured mice, tourniquets reduced tGFR by 98% and increased SUN by 825% in the NHE3Flx/Flx mice, which served as a control for NHE3 knockouts. However, tourniquets only reduced tGFR by 25% and increased SUN by 105% in the kidney epithelial cell‐specific NHE3 knockouts. The difference in the effects of tourniquets between these two strains was significant (p<0.05). Inhibition of NHE3 would reduce intracellular Na+concentrations. To determine whether intracellular Na+ regulates mitochondrial biogenesis, we treated LLC‐PK1 cells with monensin to raise intracellular Na+ levels. Monensin (10 μM) for 24 hours significantly reduced mRNA levels of PGC‐1α by 42%, NDUFS1 by 37% and ATP5o by 38% and activities of mitochondrial complex I by 100% and V by 58% in the cells. In conclusion, inhibition of NHE3 ameliorates tourniquet‐induced AKI through preservation of mitochondrial biogenesis, and this effect is most likely mediated by reducing intracellular Na+ concentration.

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.

Requirement of Na+/H+ Exchanger NHE1 for Vasopressin-Induced Osteogenic Signaling and Calcification in Human Aortic Smooth Muscle Cells

Background/Aims: Vasopressin is a powerful stimulator of vascular calcification, augmenting osteogenic signaling in vascular smooth muscle cells (VSMCs) including upregulation of transcription factors such as core-binding factor α-1 (CBFA1), msh homeobox 2 (MSX2), and SRY-Box 9 (SOX9), as well as of tissue-nonspecific alkaline phosphatase (ALPL). Vasopressin-induced osteogenic signaling and calcification require the serum- and glucocorticoid-inducible kinase 1 (SGK1). Known effects of SGK1 include upregulation of Na⁺/H⁺ exchanger 1 (NHE1). NHE1 further participates in the regulation of reactive oxygen species (ROS). NHE1 has been shown to participate in the orchestration of bone mineralization. The present study, thus, explored whether vasopressin modifies NHE1 expression and ROS generation, as well as whether pharmacological inhibition of NHE1 disrupts vasopressin-induced osteogenic signaling and calcification in VSMCs. Methods: Human aortic smooth muscle cells (HAoSMCs) were treated with vasopressin in the absence or presence of SGK1 silencing, SGK1 inhibitor GSK-650394, and NHE1 blocker cariporide. Transcript levels were determined by using quantitative real-time polymerase chain reaction, protein abundance by Western blotting, ROS generation with 2′,7′-dichlorofluorescein diacetate fluorescence, and ALP activity and calcium content by using colorimetric assays. Results: Vasopressin significantly enhanced the NHE1 transcript and protein levels in HAoSMCs, effects significantly blunted by SGK1 inhibition with GSK-650394 or SGK1 silencing. Vasopressin increased ROS accumulation, an effect significantly blocked by the NHE1 inhibitor cariporide. Vasopressin further significantly increased osteogenic markers CBFA1, MSX2, SOX9, and ALPL transcript levels, as well as ALP activity and calcium content in HAoSMCs, all effects significantly blunted by SGK1 silencing or in the presence of GSK-650394 or cariporide. Conclusion: Vasopressin stimulates NHE1 expression and ROS generation, an effect dependent on SGK1 and required for vasopressin-induced stimulation of osteogenic signaling and calcification of VSMCs.

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.

Effects of weaning on regulators of volatile fatty acid absorption and intracellular pH in Holstein calves

This study examined the effects of changes in rumen fermentation during the weaning transition on abundance of transporters involved in volatile fatty acid (VFA) absorption or intracellular pH homeostasis. Holstein bull calves (n = 27) were assigned to 1 of 3 treatment groups in a randomized, complete block design: 2 preweaning groups [animals fed milk only (PRE-M) or milk, calf starter, and hay (PRE-S)] and 1 postweaning group (animals fed milk, starter, and hay with a 2-wk weaning transition; POST-S). Calves were euthanized at 42 d of age (PRE-M and PRE-S) or at 63 d of age (POST-S), and rumen epithelium and rumen fluid samples were collected. Rumen fluid was analyzed for VFA concentration, and rumen epithelium was analyzed for the abundance of VFA transporter monocarboxylate transporter isoform 1 (MCT1) and the intracellular pH regulators sodium bicarbonate co-transporter 1 (NBC1) and sodium-proton exchanger 3 (NHE3) protein. Preweaning, total VFA concentrations tended to increase and NBC1 abundance increased with starter intake. Between pre- and postweaning, total VFA concentrations increased but NHE3 protein abundance decreased. In calves, rumen epithelial development during the weaning transition appears to show more pronounced changes in intracellular pH homeostasis than in VFA transport capacity.

Attenuating vascular stenosis-induced astrogliosis preserves white matter integrity and cognitive function

BackgroundChronic cerebral hypoperfusion (CCH) causes white matter damage and cognitive impairment, in which astrogliosis is the major pathology. However, underlying cellular mechanisms are not well defined. Activation of Na+/H+ exchanger-1 (NHE1) in reactive astrocytes causes astrocytic hypertrophy and swelling. In this study, we examined the role of NHE1 protein in astrogliosis, white matter demyelination, and cognitive function in a murine CCH model with bilateral carotid artery stenosis (BCAS).MethodsSham, BCAS, or BCAS mice receiving vehicle or a selective NHE1 inhibitor HOE642 were monitored for changes of the regional cerebral blood flow and behavioral performance for 28 days. Ex vivo MRI-DTI was subsequently conducted to detect brain injury and demyelination. Astrogliosis and demyelination were further examined by immunofluorescence staining. Astrocytic transcriptional profiles were analyzed with bulk RNA-sequencing and RT-qPCR.ResultsChronic cerebral blood flow reduction and spatial working memory deficits were detected in the BCAS mice, along with significantly reduced mean fractional anisotropy (FA) values in the corpus callosum, external capsule, and hippocampus in MRI DTI analysis. Compared with the sham control mice, the BCAS mice displayed demyelination and axonal damage and increased GFAP+ astrocytes and Iba1+ microglia. Pharmacological inhibition of NHE1 protein with its inhibitor HOE642 prevented the BCAS-induced gliosis, damage of white matter tracts and hippocampus, and significantly improved cognitive performance. Transcriptome and immunostaining analysis further revealed that NHE1 inhibition specifically attenuated pro-inflammatory pathways and NADPH oxidase activation.ConclusionOur study demonstrates that NHE1 protein is involved in astrogliosis with pro-inflammatory transformation induced by CCH, and its blockade has potentials for reducing astrogliosis, demyelination, and cognitive impairment.

Reduced activity of intestinal surface Na+/H+ exchanger NHE3 is a key factor for induction of diarrhea after PEDV infection in neonatal piglets

Porcine epidemic diarrhea virus (PEDV; family Coronaviridae, genus Alphacoronavirus) causes acute diarrhea and vomiting, dehydration, and high mortality in neonatal piglets. Despite extensive research focusing on the pathogenesis of PEDV infection, the molecular pathogenesis of PEDV-induced diarrhea in piglets remains unclear. Na+/H+ exchanger 3 (NHE3), the main exchanger of electroneutral sodium in intestinal epithelial cells, is closely associated with the occurrence of diarrhea. To date, there is no study on whether diarrhea caused by PEDV infection is related to the activity of NHE3. In the present study, it was found that the expression level of cell membrane protein NHE3 significantly decreased after PEDV infection, whereas the total level of protein expression was not significantly changed. The Na+/H+ transport rate and the mRNA abundance of NHE3 decreased; the NHE3 activity decreased gradually with increasing infection time. In vivo, after PEDV infection of newborn piglets, rupture of intestinal villi and interstitial degeneration of intestinal epithelial cells in different intestinal segments were observed by hematoxylin–eosin staining. Immunohistochemical and immunofluorescence methods were used to observe the decreased expression of NHE3 protein on the membrane of intestinal epithelial cells in the jejunum and ileum. Taken together, these data indicate that PEDV infection reduces NHE3 activity in intestinal epithelial cells, hindering Na+ transport and thus causing diarrhea.

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.

Unique Regulation of Coupled NaCl Absorption by Inducible Nitric Oxide in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Intestinal Inflammation.

In the small intestine, Na:H (NHE3) and Cl:HCO3 (DRA or PAT1) exchangers present in the brush border membrane (BBM) of absorptive villus cells are primarily responsible for the coupled absorption of NaCl, the malabsorption of which causes diarrhea, a common symptom of inflammatory bowel disease (IBD). Inducible nitric oxide (iNO), a known mediator of inflammation, is increased in the mucosa of the chronically inflamed IBD intestine. An SAMP1/YitFc (SAMP1) mouse, a spontaneous model of chronic ileitis very similar to human IBD, was used to study alterations in NaCl absorption. The SAMP1 and control AKR mice were treated with I-N(6)-(1-Iminoethyl)-lysine (L-NIL) to inhibit iNO production, and DRA/PAT1 and NHE3 activities and protein expression were studied. Though Na:H exchange activity was unaffected, Cl:HCO3 activity was significantly decreased in SAMP1 mice due to a reduction in its affinity for Cl, which was reversed by L-NIL treatment. Though DRA and PAT1 expressions were unchanged in all experimental conditions, phosphorylation studies indicated that DRA, not PAT1, is affected in SAMP1. Moreover, the altered phosphorylation levels of DRA was restored by L-NIL treatment. Inducible NO mediates the inhibition of coupled NaCl absorption by decreasing Cl:HCO3 but not Na:H exchange. Specifically, Cl:HCO3 exchanger DRA but not PAT1 is regulated at the level of its phosphorylation by iNO in the chronically inflamed intestine.

Characterization of modeled inhibitory binding sites on isoform one of the Na+/H+ exchanger

Mammalian Na+/H+ exchanger isoform one (NHE1) is a plasma membrane protein responsible for pH regulation in mammalian cells. Excess activity of the protein promotes heart disease and is a trigger of metastasis in cancer. Inhibitors of the protein exist but problems in specificity have delayed their clinical application. Here we examined amino acids involved in two modeled inhibitor binding sites (A, B) in human NHE1. Twelve mutations (Asp159, Phe348, Ser351, Tyr381, Phe413, Leu465, Gly466, Tyr467, Leu468, His473, Met476, Leu481) were made and characterized. Mutants S351A, F413A, Y467A, L468A, M476A and L481A had 40–70% of wild type expression levels, while G466A and H473A expressed 22% ~ 30% of the wild type levels. Most mutants, were targeted to the cell surface at levels similar to wild type NHE1, approximately 50–70%, except for F413A and G466A, which had very low surface targeting. Most of the mutants had measurable activity except for D159A, F413A and G466A. Resistance to inhibition by EMD87580 was elevated in mutants F438A, L465A and L468A and reduced in mutants S351A, Y381A, H473A, M476A and L481A. All mutants with large alterations in inhibitory properties showed reduced Na+ affinity. The greatest changes in activity and inhibitor sensitivity were in mutants present in binding site B which is more closely associated with TM4 and C terminal of extracellular loop 5, and is situated between the putative scaffolding domain and transport domain. The results help define the inhibitor binding domain of the NHE1 protein and identify new amino acids involved in inhibitor binding.

Interaction of NHE1 and TRPA1 Activity in DRG Neurons Isolated from Adult Rats and its Role in Inflammatory Nociception

Transient receptor potential ankyrin 1 (TRPA1) channel is expressed in a subset of nociceptive neurons. This channel integrates several nociceptive signals. Particularly, it is modulated by intracellular pH (pHi). Na+/H+ exchanger 1 (NHE1) contributes to the maintenance of pHi in nociceptors. However, it is currently unknown whether the interaction between TRPA1 and NHE1 contributes to the nociceptive processing. Thus, the purpose of this study was to assess the functional interaction between NHE1 and TRPA1 in small dorsal root ganglion (DRG) neurons from primary culture obtained from adult rats. Moreover, we also evaluated their possible interaction in acute and inflammatory pain. Zoniporide (selective NHE1 inhibitor) reduced pHi and increased intracellular calcium in a concentration-dependent fashion in DRG neurons. Zoniporide and allyl isothiocyanate (AITC, TRPA1 agonist) increased calcium transients in the same DRG neuron, whereas that A-967079 (TRPA1 antagonist) prevented the effect of zoniporide in DRG neurons. Repeated AITC induced TRPA1 desensitization and this effect was prevented by zoniporide. Both NHE1 and TRPA1 were localized at the membrane surface of DRG neurons in culture. Local peripheral zoniporide enhanced AITC-induced pronociception and this effect was prevented by A-967079. Likewise, zoniporide potentiated Complete Freund’s Adjuvant (CFA)-induced hypersensitivity, effect which was prevented by A-967079 in vivo. CFA paw injection increased TRPA1 and decresed NHE1 protein expression in DRG. These results suggest a functional interaction between NHE1 and TRPA1 in DRG neurons in vitro. Moreover, data suggest that this interaction participates in acute and inflamatory pain conditions in vivo.

Selective Renal Silencing of the Calcineurin Homologous Protein‐1 Decreases Blood Pressure in Balb/cJ Mice

Hypertension has high incidence and health care cost. Its treatments are often ineffective; therefore, we are in need of new strategies to prevent, limit or reverse hypertension. Long-term regulation of blood pressure (BP) has fundamental renal components; in particular, the causes of impaired renal Na+ handling and possible strategies to overcome disturbances in renal Na+ balance in hypertension are not well defined. Recently, we have shown that a Ca2+-binding protein, the Calcineurin Homologous Protein-1 (CHP1), is a regulatory binding partner of a key player in the control of Na+ homeostasis, the epithelial brush-border Na+/H+ exchanger-3 (NHE3). Here, we proposed that CHP1 expression controls BP as a regulatory co-factor of NHE3 transport. Indeed, selective renal silencing of Chp1 via renal subcapsular infusion of Chp1 siRNA in Balb/cJ mice significantly reduced NHE3 protein expression (-50%). Importantly, Chp1 siRNA treated mice had a reduced systolic blood pressure compared to scrambled siRNA treated mice (-30%) measured in anesthetized mice from the aorta and femoral artery, and in conscious mice using noninvasive tail-cuff method. Chp1 siRNA treated mice also had markedly reduced serum Na+ and increased urinary Na+ excretion compared to scrambled siRNA treated mice. Serum K+ and Cl- did not change while urinary K+ and Cl- excretion were increased in Chp1 siRNA treated mice. Glomerular Filtration Rate estimated by creatinine clearance was reduced in Chp1 treated mice possibly as consequence of tubuloglomerular feedback. This is the first animal model in mammals where the effect of a reduction in CHP1 protein expression in the kidney was studied. Our findings provide new insights into the action of CHP1 on renal tubular Na+ transport and propose CHP1 protein expression as a novel tool to significantly control blood pressure.

Identification, localization and expression of NHE isoforms in the alveolar epithelial cells.

Na+/H+ exchangers (NHEs), encoded by Solute Carrier 9A (SLC9A) genes in human, are ubiquitous integral membrane ion transporters that mediate the electroneutral exchange of H+ with Na+ or K+. NHEs, found in the kidney and intestine, play a major role in the process of fluid reabsorption together via Na+,K+-ATPase pump and Na+ channels. Nevertheless, the expression pattern of NHE in the lung and its role in alveolar fluid homeostasis has not been addressed. Therefore, we aimed to examine the expression of NHE specific isoforms in alveolar epithelial cells (AECs), and assess their role in congestive heart failure (CHF). Three NHE isoforms were identified in AEC and A549 cell line, at the level of protein and mRNA; NHE1, NHE2 and mainly NHE8, the latter was shown to be localized in the apical membrane of AEC. Treating A549 cells with angiotensin (Ang) II for 3, 5 and 24 hours displayed a significant reduction in NHE8 protein abundance. Moreover, the abundance of NHE8 protein was downregulated in A549 cells that were treated overnight with Ang II. NHE8 abundance in whole lung lysate was increased in rats with 1-week CHF compared to sham operated rats. However, lower abundance of NHE8 was observed in 4-week CHF group. In conclusion, we herein show for the first time, the expression of a novel NHE isoform in AEC, namely NHE8. Notably, Ang II decreased NHE8 protein levels. Moreover, NHE8 was distinctly affected in CHF rats, probably depending on the severity of the heart failure.

Long-Term Angiotensin II Infusion Induces Oxidative and Endoplasmic Reticulum Stress and Modulates Na+ Transporters Through the Nephron.

High plasma angiotensin II (Ang II) levels are related to many diseases, including hypertension, and chronic kidney diseases (CKDs). Here, we investigated the relationship among prolonged Ang II infusion/AT1 receptor (AT1R) activation, oxidative stress, and endoplasmic reticulum (ER) stress in kidney tissue. In addition, we explored the chronic effects of Ang II on tubular Na+ transport mechanisms. Male Wistar rats were subjected to sham surgery as a control or prolonged Ang II treatment (200 ng⋅kg–1⋅min–1, 42 days) with or without losartan (10 mg⋅kg–1⋅day–1) for 14 days. Ang II/AT1R induced hypertension with a systolic blood pressure of 173.0 ± 20 mmHg (mmHg, n = 9) compared with 108.0 ± 7 mmHg (mmHg, n = 7) in sham animals. Under these conditions, gene and protein expression levels were evaluated. Prolonged Ang II administration/AT1R activation induced oxidative stress and ER stress with increased Nox2, Nox4, Cyba and Ncf1 mRNA expression, phosphorylated PERK and eIF2α protein expression as well as Atf4 mRNA expression. Ang II/AT1R also raised Il1b, Nfkb1 and Acta2 mRNA expression, suggesting proinflammatory, and profibrotic effects. Regarding Na+ tubular handling, Ang II/AT1R enhanced cortical non-phosphorylated and phospho/S552/NHE3, NHE1, ENaC β, NKCC2, and NCC protein expression. Our results also highlight the therapeutic potential of losartan, which goes beyond the antihypertensive effect, playing an important role in kidney tissue. This treatment reduced oxidative stress and ER stress signals and recovered relevant parameters of the maintenance of renal function, preventing the progression of Ang II-induced CKD.