Sodium-hydrogen Exchanger Research Articles

Cytoskeletal activation of NHE1 regulates mechanosensitive cell volume adaptation and proliferation

Mammalian cells rapidly respond to environmental changes by altering transmembrane water and ion fluxes, changing cell volume. Contractile forces generated by actomyosin have been proposed to mechanically regulate cell volume. However, our findings reveal a different mechanism in adherent cells, where elevated actomyosin activity increases cell volume in normal-like cells (NIH 3T3 and others) through interaction with the sodium-hydrogen exchanger isoform 1 (NHE1). This leads to a slow secondary volume increase (SVI) following the initial regulatory volume decrease during hypotonic shock. The active cell response is further confirmed by intracellular alkalinization during mechanical stretch. Moreover, cytoskeletal activation of NHE1 during SVI deforms the nucleus, causing immediate transcriptomic changes and ERK-dependent growth inhibition. Notably, SVI and its associated changes are absent in many cancer cell lines or cells on compliant substrates with reduced actomyosin activity. Thus, actomyosin acts as a sensory element rather than a force generator during adaptation to environmental challenges.

Effectivity and safety profile of tenapanor, a sodium-hydrogen exchanger isoform 3 inhibitor, as an innovative treatment for hyperphosphatemia in chronic kidney disease: A systematic review of clinical studies

Effectivity and safety profile of tenapanor, a sodium-hydrogen exchanger isoform 3 inhibitor, as an innovative treatment for hyperphosphatemia in chronic kidney disease: A systematic review of clinical studies

The genome and comparative transcriptome of the euryhaline model ciliate Paramecium duboscqui reveal adaptations to environmental salinity

BackgroundAs a potential model organism for studies of environmental and cell biology, Paramecium duboscqui is a special euryhaline species of Paramecium that can be found in fresh, brackish, or marine water in natural salinity ranges between 0‰ and 33‰. However, the genome information as well as molecular mechanisms that account for its remarkable halotolerant traits remain extremely unknown. To characterize its genome feature, we combined PacBio and Illumina sequencing to assemble the first high-quality and near-complete macronuclear genome of P. duboscqui. Meanwhile, comparative transcriptomic profiles under different salinities gave underlying insight into the molecular mechanism of its adaptations to environmental salinity.ResultsThe results showed that the MAC genome of P. duboscqui comprises 160 contigs, with 113 of them possessing telomere (~ 28.82 Mb haploid genome size). Through comparative genomic analyses with the other ciliate, we found that gene families encoding transmembrane transporter proteins have been expanded in P. duboscqui, showing enormous potential in salinity adaptation. Like other Paramecium, P. duboscqui utilizes TGA as its only termination codon and has reassigned TAA and TAG to encode glutamine. P. duboscqui showed different growth rates under different salinities, with an optimum growth rate in 5‰ salinity. A comparison of the transcriptomic profiles among P. duboscqui grown under different concentrations showed that genes involved in protein folding, oxygen respiration, and glutathione-dependent detoxification were upregulated in the high-salt group, whereas genes encoding DNA-binding proteins and transcription factors were upregulated in the low-salt group, suggesting distinct mechanisms for responding to low and high salinity. Weighted gene coexpression network analysis (WGCNA) linked the hub genes expressed at 30‰ salinity to cysteine-type peptidase activity, lipid transfer, sodium hydrogen exchange, and cell division, with the hub genes expressed at 0‰ salinity involved in transmembrane transport and protein localization.ConclusionsThis study characterizes a new euryhaline model Paramecium, provides novel insights into Paramecium evolution, and describes the molecular mechanisms that have allow P. duboscqui to adapt to different osmotic environments.

Identification and characterization of NHX gene family for their role under salt stress in Vigna mungo.

In the current study, we have performed a comprehensive analysis of the Sodium Hydrogen Exchanger (NHX) gene family in Vigna mungo, and a total of 44 NHX genes were identified. A bimodal distribution based on domains, gene structure and phylogenetic analysis was evident. All intronpoor and intron-rich genes were clustered in clades I and II, respectively. Interestingly, all genes of subclade IIb were localized to vacuoles and possess only the NHX domain. The isoelectric point and trans-membrane domain analysis reflect the wide distribution of the NHX genes. Interestingly, Vm_NHX2 and Vm_NHX3 lacked trans-membrane domain but were found to interact with other NHX genes as well as vital salinity pathway genes, including calcium-mediated salt-responsive genes. The comparison of the mRNA sequences with that of V. marina, a halophytic species, reflects their independent evolution, majorly supporting the convergent evolution. The Ka/Ks ratio reflects the abundance of purifying selection supporting their conserved function during evolution. In our analysis, several abiotic stress and hormone-responsive elements and transcription factor binding sites were present in the promoter of the NHX genes. Further, the ion partitioning of a tolerant (K90) and a susceptible (K49) variety of V. mungo suggested that K90 managed the Na+/K+ ratio more affluently, which was also supported by profiling of superoxide radicals, hydrogen peroxide, phenol, peroxidase activity and superoxide dismutase activity. From the expression, we identified five candidate Vm_NHX genes, four of which, i.e. Vm_NHX16, Vm_NHX17, Vm_NHX29 and Vm_NHX33, were localized to the vacuolar and lysosomal membrane.

Role of NHERF1 in MicroRNA Landscape Changes in Aging Mouse Kidneys.

MicroRNAs (miRNAs) play important roles in the regulation of cellular function and fate via post-transcriptional regulation of gene expression. Although several miRNAs are associated with physiological processes and kidney diseases, not much is known about changes in miRNAs in aging kidneys. We previously demonstrated that sodium hydrogen exchanger 1 (NHERF1) expression regulates cellular responses to cisplatin, age-dependent salt-sensitive hypertension, and sodium-phosphate cotransporter trafficking. However, the mechanisms driving these regulatory effects of NHERF1 on cellular processes are unknown. Here, we hypothesize that dysregulation of miRNA-mediated gene regulatory networks that induce fibrosis and cytokines may depend on NHERF1 expression. To address this hypothesis, we compared miRNA expression in kidneys from both male and female old (12-18-month-old) and young (4-7-month-old) wild-type (WT) and NHERF1 knockout (NHERF1-/-) mice. Our results identified that miRNAs significantly decreased in NHERF1-/- mice included miR-669m, miR-590-3p, miR-153, miR-673-3p, and miR-127. Only miR-702 significantly decreased in aged WT mice, while miR-678 decreased in both WT and NHERF1-/- old versus young mice. miR-153 was shown to downregulate transcription factors NFATc2 and NFATc3 which regulate the transcription of several cytokines. Immunohistochemistry and western blotting revealed a significant increase in nuclear NFATc2 and NFATc3 in old NHERF1-/- mice compared to old WT mice. Our data further show that expression of the cytokines IL-1β, IL-6, IL-17A, MCP1, and TNF-α significantly increased in the old NHERF1-/- mice compared to the WT mice. We conclude that loss of NHERF1 expression induces cytokine expression in the kidney through interactive regulation between miR-153 and NFATc2/NFATc3 expression.

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

BackgroundDisruptions 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.MethodsAn 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.ResultsOur 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.ConclusionsThis 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.

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.

Dietary Cinnamaldehyde Activation of TRPA1 Antagonizes High-Salt-Induced Hypertension Through Restoring Renal Tubular Mitochondrial Dysfunction.

The renal proximal tubule (RPT) plays a pivotal role in regulating sodium reabsorption and thus blood pressure (BP). Transient receptor potential ankyrin 1 (TRPA1) has been reported to protect against renal injury by modulating mitochondrial function. We hypothesize that the activation of TRPA1 by its agonist cinnamaldehyde may mitigate high-salt intake-induced hypertension by inhibiting urinary sodium reabsorption through restoration of renal tubular epithelial mitochondrial function. Trpa1-deficient (Trpa1-/-) mice and wild-type (WT) mice were fed standard laboratory chow [normal diet (ND) group, 0.4% salt], standard laboratory chow with 8% salt [high-salt diet (HS) group], or standard laboratory chow with 8% salt plus 0.015% cinnamaldehyde [high-salt plus cinnamaldehyde diet (HSC) group] for 6 months. Urinary sodium excretion, reactive oxygen species (ROS) production, mitochondrial function, and the expression of sodium hydrogen exchanger isoform 3 (NHE3) and Na+/K+-ATPase of RPTs were determined. Chronic dietary cinnamaldehyde supplementation reduced tail systolic BP and 24-hour ambulatory arterial pressure in HS-fed WT mice. Compared with the mice fed HS, cinnamaldehyde supplementation significantly increased urinary sodium excretion, inhibited excess ROS production, and alleviated mitochondrial dysfunction of RPTs in WT mice. However, these effects of cinnamaldehyde were absent in Trpa1-/- mice. Furthermore, chronic dietary cinnamaldehyde supplementation blunted HS-induced upregulation of NHE3 and Na+/K+-ATPase in WT mice but not Trpa1-/- mice. The present study demonstrated that chronic activation of Trpa1 attenuates HS-induced hypertension by inhibiting urinary sodium reabsorption through restoring renal tubular epithelial mitochondrial function. Renal TRPA1 may be a potential target for the management of excessive dietary salt intake-associated hypertension.

Study of the stability of the biologically active compound N-[2-[4-oxo-3(4H)-quinazolinyl]propionyl]-guanidine using electrophoresis and mass spectrometry

At the Scientific Centre of Innovative Medicines of Volgograd State Medical University, a number of pyrimidine derivatives of guanidine were designed and synthesised to study them as promising inhibitors of sodium-hydrogen exchanger isoform 1 (NHE-1), which can reduce tissue damage during ischemia. N-[2-[4-oxo-3(4H)-quinazolinyl]propionyl]-guanidine (VMA-13-15) proved to be the most pharmacologically valuable compound. When preparing regulatory documentation for a new medicine, one of the quality criteria is the amount of related impurities. They can be technological impurities and degradation products of the active substance. The aim of the research was to study the stability of the biologically active compound N-[2-[4-[4-oxo-3(4H)-quinazolinyl]propionyl]-guanidine. Anticipated degradation products were determined by stress testing, using capillary electrophoresis and mass spectroscopy. The study of the molecule of VMA-13-15 and the use of computer technologies allowed us to predict possible degradation processes. Among them, the most likely to occur is the hydrolysis reaction during thermostating of the solution of the studied compound in an alkaline medium. In silico optimisation of the geometry of the studied structures was carried out by the MM+ molecular mechanics method using HyperChem 8.0.9. The final spatial optimisation of the geometry of the modelled molecules was carried out by the density functional theory method ub3lyp with 6-311G** basis sets. The resulting vibrational analysis of thermodynamic characteristics was carried out in Orca, which allowed us to obtain enthalpy and entropy values for the studied compounds. By capillary electrophoresis, the degradation products of an aqueous solution of the studied substance were analysed by stress tests. They were used to study the effect of thermal treatment on VMA-13-15 in the presence of 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution. We found that the alkaline environment had the greatest effect on the stability of N-[2-[4-oxo-3(4H)-quinazolinyl]propionyl]-guanidine. It was consistent with the results of the computational experiment. We studied the mass spectrum of the degradation product detected during thermostatic treatment in alkaline medium. Based on its molar mass and release time, it was confirmed that the impurity on the chromatograms of VMA-13-15 is unsubstituted quinazolin-4(3H)-one, the initial product of the synthesis

#313 Enhanced urinary phosphorous reduction: comparative study of oxylanthanum carbonate and tenapanor in rat

Abstract Background and Aims End-stage renal disease (ESRD) affects >7 M people worldwide and ∼70% of patients with ESRD have hyperphosphatemia. Oxylanthanum carbonate (OLC) is a novel nanotechnology product that combines lanthanum, which has the highest binding capacity vs other phosphate (P) binders, with a smaller pill size that is swallowed with water vs chewed. Tenapanor is a sodium hydrogen exchanger inhibitor used to reduce serum P in adults with chronic kidney disease (CKD) on dialysis. A previous study conducted by King et al found that when administered together, tenapanor and sevelamer decreased urinary p excretion significantly more than either tenapanor or sevelamer alone across all sevelamer dose levels. Employing an identical study design and dosing regimen, the objective of our study was to evaluate the effects of tenapanor and OLC on P excretion. Method In our study, we utilized the study design and dosage regimen from King et al involving sevelamer and tenapanor. 64 male Sprague Dawley rats were fed standard chow 1 week prior to study start. On study day −1, animals were randomized into 3 study groups (n = 8): 1) vehicle, 2) tenapanor 0.15 mg/kg, 3) Vehicle + OLC 0.75%, 4) Vehicle + OLC 1.5%, 5) Vehicle + OLC 3%, 6) tenapanor 0.15 mg/kg + OLC 0.75%, 7) tenapanor 0.15 mg/kg + OLC 1.5%, and 8) tenapanor 0.15 mg/kg + OLC 3%. Vehicle and tenapanor were dosed PO twice/day whereas OLC was incorporated into the diets. 24-hour urine samples were collected using metabolic cages on day 9, 10, and 11 for urinary P measurements. Results Compared to vehicle alone (group 1), P reduction of tenapanor (group 2) was 8.5 mg/day (not significant), while P reduction of OLC (group 5) was 25.3 mg/day (significant). OLC had 16.8 mg/day more P reduction than tenapanor (Fig. 1). Conclusion OLC demonstrated 3x more effective urine P reduction vs tenapanor. Subsequent analyses will focus on examining the combination of tenapanor and OLC. Combining OLC and tenapanor utilizes 2 different mechanisms of action to manage P levels while having a low pill burden, which may lead to synergistic effects to be investigated in future studies.

The salt sensitivity of Drd4-null mice is associated with the upregulations of sodium transporters in kidneys.

To explore the mechanism of the hypertension in dopamine receptor-4 (Drd4) null mice, we determined the salt sensitivity and renal sodium transport proteins in Drd4-/- and Drd4+/+ mice with varied salt diets. On normal NaCl diet (NS), mean arterial pressures (MAP, telemetry) were higher in Drd4-/- than Drd4+/+; Low NaCl diet (LS) tended to decrease MAP in both strains; high NaCl diet (HS) elevated MAP with sodium excretion decreased and pressure-natriuresis curve shifted to right in Drd4-/- relative to Drd4+/+ mice. Drd4-/- mice exhibited increased renal sodium-hydrogen exchanger 3 (NHE3), sodium-potassium-2-chloride cotransporter (NKCC2), sodium-chloride cotransporter (NCC), and outer medullary α-epithelial sodium channel (αENaC) on NS, decreased NKCC2, NCC, αENaC, and αNa+-K+-ATPase on LS, and increased αENaC on HS. NKCC2, NCC, αENaC, and αNa+-K+-ATPase in plasma membrane were greater in Drd4-/- than in Drd4+/+ mice with HS. D4R was expressed in proximal and distal convoluted tubules, thick ascending limbs, and outer medullary collecting ducts and colocalized with NKCC2 and NCC. The phosphorylation of NKCC2 was enhanced but ubiquitination was reduced in the KO mice. There were no differences between the mouse strains in serum aldosterone concentrations and urinary dopamine excretions despite their changes with diets. The mRNA expressions of renal NHE3, NKCC2, NCC, and αENaC on NS were not altered in Drd4-/- mice. Thus, increased protein expressions of NHE3, NKCC2, NCC and αENaC are associated with hypertension in Drd4-/- mice; increased plasma membrane protein expression of NKCC2, NCC, αENaC, and αNa+-K+-ATPase may mediate the salt sensitivity of Drd4-/- mice.

Defective natriuresis contributes to hyperkalemia in db/db mice during potassium supplementation.

Potassium supplementation reduces blood pressure and the occurrence of cardiovascular diseases, with K + -induced natriuresis playing a potential key role in this process. However, whether these beneficial effects occur in diabetes remains unknown. In this study, we examined the impact of high-K + intake on renal Na + /K + transport by determining the expression of major apical Na + transporters, diuretics responses (as a proxy for specific Na + transporter function), urinary Na + /K + excretion, and plasma Na + /K + concentrations in db/db mice, a model of type 2 diabetes mellitus. Although db/m mice exhibited increased fractional excretion of sodium (FE Na ) and fractional excretion of potassium (FE K ) under high-K + intake, these responses were largely blunted in db/db mice, suggesting impaired K + -induced natriuresis and kaliuresis in diabetes. Consequently, high-K + intake increased plasma K + levels in db/db mice, which could be attributed to the abnormal activity of sodium-hydrogen exchanger 3 (NHE3), sodium-chloride cotransporter (NCC), and epithelial Na + channel (ENaC), as high-K + intake could not effectively decrease NHE3 and NCC and increase ENaC expression and activity in the diabetic group. Inhibition of NCC by hydrochlorothiazide could correct the hyperkalemia in db/db mice fed a high-K + diet, indicating a key role for NCC in K + -loaded diabetic mice. Treatment with metformin enhanced urinary Na + /K + excretion and normalized plasma K + levels in db/db mice with a high-K + diet, at least partially, by suppressing NCC activity. Collectively, the impaired K + -induced natriuresis in diabetic mice under high-K + intake may be primarily attributed to impaired NCC-mediated renal K + excretion, despite the role of NHE3.

Lack of Renal NHE1 Exacerbates Lithium-Induced Nephrogenic Diabetes Insipidus

The sodium-hydrogen exchanger isoform 1 (NHE1) is a transporter with roles in transepithelial Na+/H+ transport, intracellular pH (pHi), and cell volume regulation. Within the kidney, we localized NHE1 to the basolateral membrane of the distal parts of the nephron and collecting duct (co-labelling with AQP2 for principal cells and H+-ATPase B1 subunit for all 3 types of intercalated cells: type A, B, and non-A-non-B). We have previously shown that lack of NHE3 does not affect Li+ clearance, and other studies have shown that NHE1 transports Li+ preferably compared to NHE3. Therefore we hypothesized that NHE1 plays a critical role in Li+-induced nephrogenic diabetes insipidus. To address this question, we used newly generated mice which lack NHE1 protein in the tubule/collecting duct system (NHE1KS-KO) of the kidney. After baseline measurements, control (n=12) and NHE1KS-KO (n=9) mice were switched to a Li+-containing diet (0.2% LiCl [40 mmol/kg], Harlan Teklad, TD09326) for 4 weeks. Urine osmolality was not different between groups under baseline conditions (control: 2641±178, NHE1KS-KO: 2716±244 mmol/kg). In response to Li+, urine osmolality reached a minimum after 7 days that was sustained until the end of the experimental period. Of note, urine osmolality was ~500 mmol/kg lower in NHE1KS-KO versus control mice (1125±170 versus 475±94 mmol/kg, P<0.05) despite genotypes having comparable plasma Li+ concentrations (0.48±0.1 versus 0.45±0.1 mmol/L). Plasma Na+ concentrations were not significantly different under baseline conditions (149±0.5 versus 148±0.4 mmol/L) but increased to a significantly greater extent in NHE1KS-KO versus control mice (156±1.9 versus 152±0.5 mmol/L, P<0.05). Along those lines, plasma Cl− was significantly greater in NHE1KS-KO versus control mice at the end of the experimental period (117±1.9 versus 111±0.4 mmol/L, P<0.05). Taken together, our data indicate that NHE1 in principal cells plays an important role in the development of nephrogenic diabetes insipidus as well as the development of hypernatremia and hyperchloremia. This work was supported by a VA Merit Review Award IBX004968A (to Dr. Rieg) and a Pilot Project from the USF Microbiomes Institute (to T.R. and J.D.R). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nascent shifts in renal cellular metabolism, structure, and function due to chronic empagliflozin in prediabetic mice.

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

Antiarrhythmic Effect of SGLT-2 Inhibitors in high-degree AV Block Caused by Heart Failure: A Case Report

Highlights: 1. The antiarrhythmic effect of SGLT-2 inhibitors, particularly empagliflozin, is a very interesting topic to discuss further. 2. In patients with high degree AV block due to HF in rural areas, the use of SGLT-2 inhibitors is quite rare in Indonesia. - Background: According to WHO's Top 10 Global Health Threats, non-communicable diseases such as heart failure contribute to more than 70% of all deaths worldwide. Several studies have shown that SGLT-2 inhibitors can considerably reduce HF. As research progresses, SGLT-2 inhibitors may lessen the risk of arrhythmias in HF patients. Case Summary: A 57-year-old female with uncontrolled hypertension came to the ED after collapsing at home. The vital signs and physical examination are normal. The preliminary ECG showed a high degree AV Block with multiple multifocal PVC. Early laboratory revealed mild hypokalemia. The patient experienced a seizure the next day, and an ECG showed total AV block with a non-sustained VT episode with PVC R on T. Following critical cardiac care and stabilization, the patient underwent echocardiography, which showed mild MR and AR with a reduced ejection fraction (47.7%). Once the patient's condition has stabilized, the patient is given 1x10mg Empagliflozin PO. Discussion: SGLT-2 inhibitors are beneficial in the treatment of heart failure and arrhythmia. SGLT-2 inhibitors have antiarrhythmic effects through a variety of pathways, such as lowering preload and afterload, inhibition of sodium-hydrogen exchange in myocardial cells, and suppression of the sympathetic nervous system.

Empagliflozin mitigates cardiac hypertrophy through cardiac RSK/NHE-1 inhibition

BackgroundSGLT2i reduce cardiac hypertrophy, but underlying mechanisms remain unknown. Here we explore a role for serine/threonine kinases (STK) and sodium hydrogen exchanger 1(NHE1) activities in SGLT2i effects on cardiac hypertrophy. MethodsIsolated hearts from db/db mice were perfused with 1 µM EMPA, and STK phosphorylation sites were examined using unbiased multiplex analysis to detect the most affected STKs by EMPA. Subsequently, hypertrophy was induced in H9c2 cells with 50 µM phenylephrine (PE), and the role of the most affected STK (p90 ribosomal S6 kinase (RSK)) and NHE1 activity in hypertrophy and the protection by EMPA was evaluated. ResultsIn db/db mice hearts, EMPA most markedly reduced STK phosphorylation sites regulated by RSKL1, a member of the RSK family, and by Aurora A and B kinases. GO and KEGG analysis suggested that EMPA inhibits hypertrophy, cell cycle, cell senescence and FOXO pathways, illustrating inhibition of growth pathways. EMPA prevented PE-induced hypertrophy as evaluated by BNP and cell surface area in H9c2 cells. EMPA blocked PE-induced activation of NHE1. The specific NHE1 inhibitor Cariporide also prevented PE-induced hypertrophy without added effect of EMPA. EMPA blocked PE-induced RSK phosphorylation. The RSK inhibitor BIX02565 also suppressed PE-induced hypertrophy without added effect of EMPA. Cariporide mimicked EMPA’s effects on PE-treated RSK phosphorylation. BIX02565 decreased PE-induced NHE1 activity, with no further decrease by EMPA. ConclusionsRSK inhibition by EMPA appears as a novel direct cardiac target of SGLT2i. Direct cardiac effects of EMPA exert their anti-hypertrophic effect through NHE-inhibition and subsequent RSK pathway inhibition.

Small Molecules for the Treatment of Long-COVID-Related Vascular Damage and Abnormal Blood Clotting: A Patent-Based Appraisal.

People affected by COVID-19 are exposed to, among others, abnormal clotting and endothelial dysfunction, which may result in deep vein thrombosis, cerebrovascular disorders, and ischemic and non-ischemic heart diseases, to mention a few. Treatments for COVID-19 include antiplatelet (e.g., aspirin, clopidogrel) and anticoagulant agents, but their impact on morbidity and mortality has not been proven. In addition, due to viremia-associated interconnected prothrombotic and proinflammatory events, anti-inflammatory drugs have also been investigated for their ability to mitigate against immune dysregulation due to the cytokine storm. By retrieving patent literature published in the last two years, small molecules patented for long-COVID-related blood clotting and hematological complications are herein examined, along with supporting evidence from preclinical and clinical studies. An overview of the main features and therapeutic potentials of small molecules is provided for the thromboxane receptor antagonist ramatroban, the pan-caspase inhibitor emricasan, and the sodium-hydrogen antiporter 1 (NHE-1) inhibitor rimeporide, as well as natural polyphenolic compounds.

Myometrium infection decreases TREK1 through NHE1 and increases contraction in pregnant mice.

Intrauterine infection during pregnancy can enhance uterine contractions. A two-pore K+ channel TREK1 is crucial for maintaining uterine quiescence and reducing contractility, with its properties regulated by pH changes in cell microenvironment. Meanwhile, the sodium hydrogen exchanger 1 (NHE1) plays a pivotal role in modulating cellular pH homeostasis, and its activation increases smooth muscle tension. By establishing an infected mouse model of Escherichia coli (E. coli) and lipopolysaccharide (LPS), we used Western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence to detect changes of TREK1 and NHE1 expression in the myometrium, and isometric recording measured the uterus contraction. The NHE1 inhibitor cariporide was used to explore the effect of NHE1 on TREK1. Finally, cell contraction assay and siRNA transfection were performed to clarify the relationship between NHE1 and TREK1 in vitro. We found that the uterine contraction was notably enhanced in infected mice with E. coli and LPS administration. Meanwhile, TREK1 expression was reduced, whereas NHE1 expression was upregulated in infected mice. Cariporide alleviated the increased uterine contraction and promoted myometrium TREK1 expression in LPS-injected mice. Furthermore, suppression of NHE1 with siRNA transfection inhibited the contractility of uterine smooth muscle cells and activated the TREK1. Altogether, our findings indicate that infection increases the uterine contraction by downregulating myometrium TREK1 in mice, and the inhibition of TREK1 is attributed to the activation of NHE1.NEW & NOTEWORTHY Present work found that infection during pregnancy will increase myometrium contraction. Infection downregulated NHE1 and followed TREK1 expression and activation decrease in myometrium, resulting in increased myometrium contraction.

N123I mutation in the ALV-J receptor-binding domain region enhances viral replication ability by increasing the binding affinity with chNHE1.

The subgroup J avian leukosis virus (ALV-J), a retrovirus, uses its gp85 protein to bind to the receptor, the chicken sodium hydrogen exchanger isoform 1 (chNHE1), facilitating viral invasion. ALV-J is the main epidemic subgroup and shows noteworthy mutations within the receptor-binding domain (RBD) region of gp85, especially in ALV-J layer strains in China. However, the implications of these mutations on viral replication and transmission remain elusive. In this study, the ALV-J layer strain JL08CH3-1 exhibited a more robust replication ability than the prototype strain HPRS103, which is related to variations in the gp85 protein. Notably, the gp85 of JL08CH3-1 demonstrated a heightened binding capacity to chNHE1 compared to HPRS103-gp85 binding. Furthermore, we showed that the specific N123I mutation within gp85 contributed to the enhanced binding capacity of the gp85 protein to chNHE1. Structural analysis indicated that the N123I mutation primarily enhanced the stability of gp85, expanded the interaction interface, and increased the number of hydrogen bonds at the interaction interface to increase the binding capacity between gp85 and chNHE1. We found that the N123I mutation not only improved the viral replication ability of ALV-J but also promoted viral shedding in vivo. These comprehensive data underscore the notion that the N123I mutation increases receptor binding and intensifies viral replication.

Effect of an NHE3 inhibitor in combination with an NPT2b inhibitor on gastrointestinal phosphate absorption in Rodent models.

Many of the pathological consequences of chronic kidney disease can be attributed to an elevation in serum phosphate levels. Current therapies focused on decreasing intestinal phosphate absorption to treat hyperphosphatemia are inadequate. The most effective therapeutic strategy may be to target multiple absorptive pathways. In this study, the ability of a novel inhibitor of the intestinal sodium hydrogen exchanger 3 (NHE3), LY3304000, which inhibits paracellular, diffusional uptake of phosphate, to work in combination with an inhibitor of the active transporter, sodium dependent phosphate cotransporter 2b (NPT2b), LY3358966, was explored. LY3304000 modestly inhibited the acute uptake of phosphate into plasma of rats, while surprisingly, it doubled the rate of phosphate uptake in mice, an animal model dominated by NPT2b mediated acute phosphate uptake. In rats, LY3004000 and LY3358966 work in concert to inhibit acute phosphate uptake. On top of LY3358966, LY3304000 further decreased the acute uptake of phosphate into plasma. Studies measuring the recovery of radiolabeled phosphate in the intestine demonstrated LY3304000 and LY3358966 synergistically inhibited the absorption of phosphate in rats. We hypothesize the synergism is because the NHE3 inhibitor, LY3304000, has two opposing effects on intestinal phosphate absorption in rats, first it decreases diffusion mediated paracellular phosphate absorption, while second, it simultaneously increases phosphate absorption through the NPT2b pathway. NHE3 inhibition decreases proton export from enterocytes and raises the cell surface pH. In vitro, NPT2b mediated phosphate transport is increased at higher pHs. The increased NPT2b mediated transport induced by NHE3 inhibition is masked in rats which have relatively low levels of NPT2b mediated phosphate transport, by the more robust inhibition of diffusion mediated phosphate absorption. Thus, the inhibition of NPT2b mediated phosphate transport in rats in the presence of NHE3 inhibition has an effect that exceeds its effect in the absence of NHE3 inhibition, leading to the observed synergism on phosphate absorption between NPT2b and NHE3 inhibition.