Sodium Gradient Research Articles

Does a transmembrane sodium gradient control membrane potential in mammalian mitochondria?

In a recent publication, Hernansanz-Agusti̒n et al. propose that a sodium gradient across the inner mitochondrial membrane, generated by a Na+/H+ activity integral to Complex I can account for half of the mitochondrial membrane potential. This conflicts with conventional electrophysiological and chemiosmotic understanding.

Transport and inhibition mechanisms of the human noradrenaline transporter.

The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons1-3. It is a pharmacological target for various antidepressants and analgesic drugs4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.

Blocking Aδ- and C-fiber neural transmission by sub-kilohertz peripheral nerve stimulation.

We recently showed that sub-kilohertz electrical stimulation of the afferent somata in the dorsal root ganglia (DRG) reversibly blocks afferent transmission. Here, we further investigated whether similar conduction block can be achieved by stimulating the nerve trunk with electrical peripheral nerve stimulation (ePNS). We explored the mechanisms and parameters of conduction block by ePNS via ex vivo single-fiber recordings from two somatic (sciatic and saphenous) and one autonomic (vagal) nerves harvested from mice. Action potentials were evoked on one end of the nerve and recorded on the other end from teased nerve filaments, i.e., single-fiber recordings. ePNS was delivered in the middle of the nerve trunk using a glass suction electrode at frequencies of 5, 10, 50, 100, 500, and 1000 Hz. Suprathreshold ePNS reversibly blocks axonal neural transmission of both thinly myelinated Aδ-fiber axons and unmyelinated C-fiber axons. ePNS leads to a progressive decrease in conduction velocity (CV) until transmission blockage, suggesting activity-dependent conduction slowing. The blocking efficiency is dependent on the axonal conduction velocity, with Aδ-fibers efficiently blocked by 50-1000 Hz stimulation and C-fibers blocked by 10-50 Hz. The corresponding NEURON simulation of action potential transmission indicates that the disrupted transmembrane sodium and potassium concentration gradients underly the transmission block by the ePNS. The current study provides direct evidence of reversible Aδ- and C-fiber transmission blockage by low-frequency (<100 Hz) electrical stimulation of the nerve trunk, a previously overlooked mechanism that can be harnessed to enhance the therapeutic effect of ePNS in treating neurological disorders.

First-In-Man Trial of β3-Adrenoceptor Agonist Treatment in Chronic Heart Failure: Impact on Diastolic Function.

Diastolic dysfunction (DD) in heart failure is associated with increased myocardial cytosolic calcium and calcium-efflux through the sodium-calcium exchanger depends on the sodium gradient. Beta-3-adrenoceptor (β3-AR) agonists lower cytosolic sodium and have reversed organ congestion. Accordingly, β3-AR agonists might improve diastolic function, which we aimed to assess. In a first-in-man, randomized, double-blinded trial, we assigned 70 patients with HF with reduced ejection fraction, New York Heart Association II-III, and left ventricular ejection fraction <40% to receive the β3-AR agonist mirabegron (300 mg/day) or placebo for 6 months, in addition to recommended heart failure therapy. We performed echocardiography and cardiac computed tomography and measured N-terminal probrain natriuretic peptide at baseline and follow-up. DD was graded per multiple renowned algorithms. Baseline and follow-up data were available in 57 patients (59 ± 11 years, 88% male, 49% ischemic heart disease). No clinically significant changes in diastolic measurements were found within or between the groups by echocardiography (E/e' placebo: 13 ± 7 to 13 ± 5, P = 0.21 vs. mirabegron: 12 ± 6 to 13 ± 8, P = 0.74, between-group follow-up difference 0.2 [95% CI, -3 to 4], P = 0.89) or cardiac computed tomography (left atrial volume index: between-group follow-up difference 9 mL/m 2 [95% CI, -3 to 19], P = 0.15). DD gradings did not change within or between the groups following 2 algorithms ( P = 0.72, P = 0.75). N-terminal probrain natriuretic peptide remained unchanged in both the groups ( P = 0.74, P = 0.64). In patients with HF with reduced ejection fraction, no changes were identified in diastolic measurements, gradings or biomarker after β3-AR stimulation compared with placebo. The findings add to the previous literature questioning the role of impaired Na + -Ca 2+ -mediated calcium export as a major culprit in DD. NCT01876433.

Redefining the concept of residual renal function with kidney sodium MRI: a pilot study.

The concept of residual kidney function (RKF) is exclusively based upon urine volume and small solute clearance, making RKF challenging to assess in clinical practice. The aim of this study was to test the technical feasibility of obtaining useable 23Na-MRI kidney images in hemodialysis (HD) participants. We conducted an exploratory prospective study to quantify the cortico-medullary sodium gradient in healthy and HD participants. Participants fasted for eight hours prior to their study visit. Urine samples were collected to measure urinary osmolarity, before MRI. Proton and sodium pictures were merged; ROIs were delineated for the medulla and cortex when feasible. In cases where cortex could not be identified, we considered the cortico to medulla gradient (CMG) to be no longer present, resulting in a medulla-to-cortex ratio of 1. 17 healthy volunteers and 21 HD participants. Median (IQR) fasting medulla to cortex ratio was significantly higher 1.56 [1.5-1.61] in healthy volunteers compared to HD patients 1.22 [1.13-1.3], p<0.0001. Medulla to cortex ratio and median urinary osmolarity were correlated (r=0.87, p<0.0001) in the whole population. We found a significant association between HD vintage and medulla to cortex ratio whereas we did not find any association with urine volume. Sodium signal intensity distribution within healthy kidney describes two different peaks- relating to well defined cortex and medulla; whereas HD participants displays only a single peak indicative of the markedly lower sodium concentration. This study is only an exploratory study with a modest number of patients. the application of kidney sodium MRI to the study of RKF in patients receiving maintenance HD is practical and provides a previously unavailable ability to interrogate the function of remnant tubular function.

Does the concentration ratio contribute to the scaling of circulating cooling seawater?

Due to the impact of freshwater shortage on coastal areas, coastal factories have gradually chosen seawater circulation cooling towers for industrial cooling. The circulating seawater easily generates thick scales, obstructs pipes, and affects heat transfer. Different from the development of scale inhibitors and membrane treatment, there are few fundamental researches on scale formation in seawater pretreatment. In this study, the effects of seawater concentration on scaling time and crystal shapes were investigated through on-line monitoring scaling experiments and scale collection. When the concentration of seawater increased (0.5–2.5 times), the scaling time was delayed, and the formation of large aggregates decreased. Subsequently, the scale delay of calcium carbonate caused by high ion concentration was confirmed by sodium and potassium ion gradient tests and the Visual MINTEQ model. The dynamic simulation further revealed that high ion saturation inhibits calcium and carbonate ions' collision, binding, and agglomeration. Sodium ions inhibit scale formation through electrostatic repulsion with calcium ions, while potassium ions occupy spatial sites around calcium ions. Furthermore, the aragonite formation tendency in highly ion-saturated solutions provides a theoretical basis for industrial carbon capture technology at the end of cyclic cooling.

The effects of semaglutide, empagliflozin and their combination on the kidney sodium signal from magnetic resonance imaging: A prespecified, secondary analysis from a randomized, clinical trial

AimsTo evaluate the effect of treatment with semaglutide and empagliflozin on the cortico-medullary sodium gradient (MCR; medulla/cortex ratio), urine sodium/creatinine ratio (UNACR), and estimated plasma volume (ePV) and to compare the MCR between persons with and without type 2 diabetes. MethodsUsing the 23Na magnetic resonance imaging (23Na-MRI) technique, we investigated the effects of 32 weeks of treatment with semaglutide, empagliflozin or their combination on MCR in 65 participants with type 2 diabetes and high risk of cardiovascular disease. The participants were recruited from a randomized, controlled interventional trial and further characterized by UNACR and ePV. In addition, in a cross-sectional design, we compared MCR by 23Na-MRI in 12 persons with type 2 diabetes and 17 matched controls. Data from the interventional trial were analyzed using a single, multivariate linear mixed model strategy for repeated measurements. Data from the cross-sectional study were analyzed by fitting a linear regression model adjusted for age and sex. ResultsCompared to placebo, semaglutide, but not empagliflozin, significantly decreased the MCR (−9 %, 95%CI (−18, −0.06)%, p = 0.035 and −0.05 %, 95%CI(−0.15, 0.05)%, p = 0.319, respectively). The UNACR decreased in the semaglutide group(−35 %, 95 % CI(−52, −14) %, p = 0.003) but not in the empagliflozin group (7 %, 95 % CI(−21, 44)%, p = 0.657), whereas the ePV decreased in the combination group. The MCR was not different between persons with and without type 2 diabetes. Conclusion23Na magnetic resonance imaging can identify drug induced changes in the MCR in persons with type 2 diabetes, and 32 weeks of semaglutide decreases the MCR in such persons. There is no difference in the MCR between persons with and without type 2 diabetes. Trial number and registryEUDRACT 2019-000781-38, clinicaltrialsregister.eu.

Association of Bioimpedance Parameters with Increases in Blood Pressure during Hemodialysis.

Intradialytic hypertension, defined as an increase in BP from pre- to post-hemodialysis (HD), affects 5%-15% of patients receiving maintenance HD and is associated with cardiovascular and all-cause mortality. Hypervolemia is believed to be a major etiological factor, yet the association of more objective biomarkers of volume status with intradialytic hypertension is not well described. In a post hoc analysis of the Frequent Hemodialysis Network Daily Trial ( n =234), using data from baseline, 1-, 4-, and 12-month visits ( n =800), we used random-effects regression to assess the association of bioimpedance estimates of volume (vector length) with post-HD systolic BP (continuous) and any increase in systolic BP (categorical) from pre- to post-HD. We adjusted models for randomized group; age; sex; self-reported race; Quételet (body mass) index; vascular access; HD vintage; hypertension; history of heart failure; diabetes; residual kidney function (urea clearance); pre-HD systolic BP; ultrafiltration rate; serum-dialysate sodium gradient; and baseline values of hemoglobin, phosphate, and equilibrated Kt/V urea. The mean age of participants was 50±14 years, 39% were female, and 43% were Black. In adjusted models, shorter vector length (per 50 Ω/m) was associated with higher post-HD systolic BP (2.9 mm Hg; 95% confidence interval [CI], 1.6 to 4.3) and higher odds of intradialytic hypertension (odds ratio 1.66; 95% CI, 1.07 to 2.55). Similar patterns of association were noted with a more stringent definition of intradialytic hypertension (>10 mm Hg increase from pre- to post-HD systolic BP), where shorter vector length (per 50 Ω/m) was associated with a higher odds of intradialytic hypertension (odds ratio 2.17; 95% CI, 0.88 to 5.36). Shorter vector length, a bioimpedance-derived proxy of hypervolemia, was independently associated with higher post-HD systolic BP and risk of intradialytic hypertension.

Conformational coupling of redox-driven Na+-translocation in Vibrio cholerae NADH:quinone oxidoreductase

In the respiratory chain, NADH oxidation is coupled to ion translocation across the membrane to build up an electrochemical gradient. In the human pathogen Vibrio cholerae, the sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) generates a sodium gradient by a so far unknown mechanism. Here we show that ion pumping in Na+-NQR is driven by large conformational changes coupling electron transfer to ion translocation. We have determined a series of cryo-EM and X-ray structures of the Na+-NQR that represent snapshots of the catalytic cycle. The six subunits NqrA, B, C, D, E, and F of Na+-NQR harbor a unique set of cofactors that shuttle the electrons from NADH twice across the membrane to quinone. The redox state of a unique intramembranous [2Fe-2S] cluster orchestrates the movements of subunit NqrC, which acts as an electron transfer switch. We propose that this switching movement controls the release of Na+ from a binding site localized in subunit NqrB.

Ian Michael Glynn. 3 June 1928—7 July 2022

In the mid 1950s the convergence of crucial discoveries on nerve and muscle physiology brought to centre stage the question of how sodium and potassium concentration gradients needed for nerve conduction and muscle contraction are maintained. This was the subject Ian Glynn set out to investigate when he returned to Cambridge as a Trinity College graduate student in 1953. His initial results on the sodium and potassium permeabilities of the human red blood cell membrane, published in 1956–1967, established these cells as an ideal experimental system for studying the transport properties of the ‘sodium pump’ because of the ease with which the ionic composition of intra- and extracellular environments could be controlled, cell metabolism managed and ionic fluxes measured using isotopes. This work was followed by ground-breaking contributions from his research group over the next decades in a long series of brilliantly designed experiments, setting the foundations of our current understanding of the molecular mechanism of the sodium/potassium pump. Ian became a Trinity College fellow in 1960, advanced rapidly to a professorship in the Department of Physiology after being elected Fellow of the Royal Society in 1970, and was appointed head of department in 1986 until his retirement in 1995.

#3528 DIALYSATE SODIUM: IS IT STILL MYTH OR NOT?

Abstract Background and Aims Pre-hemodialysis serum sodium levels can vary among patients, therefore, a single dialysate sodium prescription may not be appropriate for all patients. The aim of the study was to investigate whether dialysis patients will have some beneficial effects of prescription of different models of dialysate sodium. Method 77 nondiabetic subjects performed 12 months hemodialysis (HD) sessions with dialysate sodium concentration set up at 138 mmol/L, followed by additional 4 models of dialysate sodium (each one 4 months sessions lasted) wherein dialysate sodium was set up: model 1: according to pre-hemodialysis serum sodium concentration, model 2: sodium concentration in UF fluid, model 3: sodium profiling (144-136 mmol/L, every hour decreasing of dialysate sodium during dialysis), model 4: stepwise dialysate sodium (starting dialysis with a dialysate sodium of 138 mmol/L and reducing it every week by 1 mmol/L sodium for the next 4 weeks). Blood pressure (BP), interdialytic weight gain (IDWG), thirst score, sodium gradient were analysed. After the standard dialysate sodium hemodialyses, the subjects were divided into normotensive, hypertensive and hypotensive based on the average pre-hemodialysis systolic BP during the standard dialysate sodium hemodialyses. Results Model 1: resulted in significantly lower BP (153.60±14.26 versus 133,61±11.88 mmHg; p = 0.000) and IDWG (2.21±0.93 versus 1.87±0.92 kg; p = 0.018) in hypertensive patients, whereas normotensive patients showed only significant decrease in IDWG (p = 0,004). Hypertensive patients had significant highest sodium gradient compared to other patients (p&amp;lt;0.05), followed by significant increase of 0,6% IDWG confirmed with univariate regression analysis. Thirst score was significantly lower in all patients with individualized-sodium HD and no relationship between the sodium gradient and the subjective feeling of thirst was confirmed (p = 0.368). Model 2: resulted in significantly lower BP in normotensive and hypertensive patients, with no influence on IDWG and thirst score compared to standard dialysate sodium (p = 0.474 and p = 0.212, retrospectively). Patients who were hypotensive, due to frequent collapses and unmeasurable blood pressure values, as well as for ethical reasons, were excluded from analysis with this dialysate sodium model. Model 3: confirmed significantly higher BP and IDWG in all 3 groups (p = 0.006) and significantly higher thirst score in normo and hipotensive patients (p = 0.000), with no influence in hypertensive patients. Model 4: significant decrease in SBP and DBP in hypertensive patients and insignificant increase in IDWG, but with no effects in normotensive and hypotensive patients on BP, IDWG and thirst score. Conclusion Model 1 resulted in better clinical outcome in hypertensive and normotensive patients compared to standard dialysate sodium, whereas other 3 models didn't show any clinical benefits.

#6844 MONITOR SWITCHING-INDUCED VARIATIONS IN CONDUCTIVITY AND PLASMA SODIUM DURING HEMODIALYSIS SESSIONS

Abstract Background and Aims Until the advent of the new monitors with sodium modules, the differences between prescribed and actual sodium have been little studied. And most dialysis centers have a fixed sodium prescription. However, the sodium gradient during sessions is one of the key factors in its balance in hemodialysis patients. Predialysis plasma sodium has an important interindividual but not intraindividual variability, which supports the hypothesis of reference sodium (setpoint). This work aimed to compare the impact of switching from the 5008 Cordiax monitor to the new 6008 Cordiax monitor on the actually measured conductivity and initial and final plasma sodium. Method A total of 106 hemodialysis patients were included. Each patient received two dialysis sessions in which only the monitor was varied. The variables collected were: concentrate, prescribed sodium and bicarbonate, real conductivity, initial and final plasma sodium measured by ionic dialysance, and the change in plasma sodium concentration during treatment (ΔPNa), which has been related to mortality when it exceeds 4 mmol/L, was calculated. The clinical research ethics committee of Hospital Clínic de Barcelona approved the present study. Results The change of dialysis monitor showed small, although significant, differences in initial (138.14 mmol/L with 5008 vs. 138.81 mmol/L with 6008) and final plasma sodium (139.58 mmol/L vs. 140.97 mmol/L), as well as in the actual conductivity obtained (13.97 vs. 14.1 mS/cm). The ΔPNa also increased significantly, with the percentage of patients with a ΔPNa greater than 4 mmol/L going from 6 to 22% with the monitor change. All these differences were statistically significant regardless of the concentrate used. All sessions were performed without notable clinical incidents, hypotension, cramps or thirst episodes. Conclusion The ultimate goal of hemodialysis should be to achieve isonatremia that allows complete elimination of interdialytic sodium gain and avoid intradialytic sodium loading. The change from 5008 to 6008 monitor is associated with an increase in conductivity, higher plasma sodium, and an increase in ΔPNa. Knowing and confirming this change will allow us to individualize the sodium prescription to find adequate isonatremic dialysis and, thus, avoid possible adverse effects.

#6780 EFFECTS OF ISONATREMIC DIALYSIS ON INTERDIALYTIC WEIGHT GAIN RELYING ON AN AUTOMATED SODIUM CONTROL ALGORITHM: INTERIM ANALYSIS OF DISON-IRC STUDY

Abstract Background and Aims Sodium (Na) is a major determinant of extracellular fluid volume and osmolarity homeostasis. Chronic fluid overload (FO) is present in 30 to 50% of hemodialysis patients (HD). HD patients are mostly treated with fixed dialysate Na concentration irrespective of plasma Na concentration. This fixed approach tends to displace patient's osmolar set point and stimulates thirst and weight gain. An automated personalized dialysate Na alignment to patient's natremia to avoid unwanted diffusive Na gain and tonicity change induced by HD seems preferable. Several studies exploring individualized dialysate Na prescription have reported inconsistent results. In this prospective interventional multicenter study, we aimed to explore the impact of dialysate Na individualization through isonatremic HD on interdialytic weight gain (IDWG) (primary outcome) and dialytic HD-related symptoms (secondary outcome). For this purpose, we have used the automated “Na Control” option for adjusting dialysate Na to minimize dialysate-plasma sodium gradient and deliver an isonatremic dialysis. Method This is a longitudinal study comparing 2 periods: Period-1 = observational phase (2 months) → Period-2 = isonatremic phase by activation of “Na Control” in isonatremic mode (2 months) → comparison of IDWG between both periods. The embedded “sodium control” option in HD machine was used in all cases of Period-2 to actively align dialysate sodium concentration to natremia and quantify net sodium mass transfer. Results are expressed as mean±SD or median [25th-75th]. Results We present preliminary results obtained through 6 dialysis facilities including 51 patients, 33 (64.7%) females, 75 [65-81] years old, dry weight 71.9 [60.5-78.0] kg. Main findings are as follows: Firstly, an excellent agreement was observed between plasma Na concentrations, measured (laboratory) and estimated (machine) from HD monitor (mean difference before dialysis: 0.05, after dialysis: 0.62); Secondly, plasma Na estimated was as follows: before dialysis plasma Na (137.6 [136.2-139.0] vs 137.2 [136.0-139.3] and after dialysis plasma Na (139.0 [137.6-139.7] vs 137.1 [135.7-138.9]) in Period-1 and Period-2 respectively; Thirdly, the primary outcome (IDWG) as well as the diffusive Na balance were significantly reduced by isonatremic dialysis condition (IDWG: 2.17±0.92 versus 1.95±0.86 kg, p&amp;lt;0,0001; diffusive Na balance (3.9 [1.9-5.1] versus 0.7 [0.0-2.7] g of Na, p&amp;lt;0.0001). Conclusion Isonatremic dialysis condition automatically driven by “Na control” embedded In HD monitor was confirmed safe and easily implemented in clinical workflow. In addition, these preliminary results show that isonatremic dialysis improved dialysis patient perception and significantly reduced IDWG.

Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus.

A genome-scale metabolic model, encompassing a total of 623 genes, 727 reactions, and 865 metabolites, was developed for Pyrococcus furiosus, an archaeon that grows optimally at 100°C by carbohydrate and peptide fermentation. The model uses subsystem-based genome annotation, along with extensive manual curation of 237 gene-reaction associations including those involved in central carbon metabolism, amino acid metabolism, and energy metabolism. The redox and energy balance of P. furiosus was investigated through random sampling of flux distributions in the model during growth on disaccharides. The core energy balance of the model was shown to depend on high acetate production and the coupling of a sodium-dependent ATP synthase and membrane-bound hydrogenase, which generates a sodium gradient in a ferredoxin-dependent manner, aligning with existing understanding of P. furiosus metabolism. The model was utilized to inform genetic engineering designs that favor the production of ethanol over acetate by implementing an NADPH and CO-dependent energy economy. The P. furiosus model is a powerful tool for understanding the relationship between generation of end products and redox/energy balance at a systems-level that will aid in the design of optimal engineering strategies for production of bio-based chemicals and fuels. IMPORTANCE The bio-based production of organic chemicals provides a sustainable alternative to fossil-based production in the face of today's climate challenges. In this work, we present a genome-scale metabolic reconstruction of Pyrococcus furiosus, a well-established platform organism that has been engineered to produce a variety of chemicals and fuels. The metabolic model was used to design optimal engineering strategies to produce ethanol. The redox and energy balance of P. furiosus was examined in detail, which provided useful insights that will guide future engineering designs.

Maintaining and Restoring Gradients of Ions in the Epidermis: The Role of Ion and Water Channels in Acute Cutaneous Wound Healing.

Significance: Aquaporins and ion channels establish and regulate gradients of calcium, sodium, potassium, chloride, water, and protons in the epidermis. These elements have been found to play significant roles in skin biology and wound healing. In this study, we review our understanding of these channels and ion gradients, with a special emphasis on their role in acute wound healing. Recent Advances: Specifically, we assess the temporal and spatial arrangements of ions and their respective channels in the intact skin and during wound and healing to provide a novel perspective of the role of ionic gradients through the various stages of wound healing. Critical Issues: The roles of gradients of ions and channels in wound healing are currently not well understood. A collective analysis of their traits and arrangements in the skin during wound healing may provide a new perspective and understanding of the functionality of gradients of ions and channels in skin biology and wound healing. Future Directions: It is important to elucidate how the gradients of ions and ion channels regulate and facilitate wound healing. A better understanding of the ionic environments may identify novel therapeutic targets and improved strategies to promote wound healing and possibly treat other cutaneous diseases.

Investigation of the transport cycle of sodium glucose transport proteins through computational simulation.

The human Sodium-Glucose Linked Transporter hSGLT2 is responsible for the reabsorption of sugar in the kidneys, and as such is a key target for the treatment of type 2 diabetes. SGLTs are found in the cell membrane and use the sodium gradient to transport sugar into cells through an alternating-access mechanism. Experimentally derived structures of the bacterial homolog vSGLT in an inward-open conformation (apo and substrate bound) and the related sialic acid transporter in an outward-open conformation, along with more recent structures of partially inward-open hSGLT1 and partially outward-open inhibitor-bound hSGLT2, give some insight to the transport mechanism. However, the exact nature of the conformational transition and the energetic details that govern the transport cycle remain unclear. We have used molecular dynamics simulations to explore the dynamics of SGLT in the presence or absence of substrate, including unbiased simulations in different conformations, and weighted ensemble simulations using WESTPA to explore the conformational transition landscape. These simulations demonstrate how the presence of substrate can alter the energetics of the conformational landscape; for example, how sugar may lock the outer gate closed, preventing substrate leaks. Improving our understanding of SGLT function through this work is a key step to better inform the treatment of diabetes and other SGLT-related diseases.

Biochemical and molecular characterization of the most-common SLC13A5-Epilepsy causing missense-mutations.

The sodium-coupled citrate transporter (NaCT) is a plasma membrane transporter, which is energized by an inwardly directed electrochemical sodium gradient. It mediates the symport of sodium and the carboxylate citrate into cells. NaCT is expressed in the liver, testis, brain, bone, and teeth, where citrate plays key roles in the synthesis of neurotransmitters, cholesterol, and fatty acids, the generation of energy, and teeth/bone mineralization. In humans, loss-of-function mutations in SLC13A5, the NaCT gene, cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5-Epilepsy), which leads to epilepsy, impaired speech, limited motor skills, developmental delay, and tooth defects. Currently, there is no treatment for EIEE25. Recently, the cryo-electron microscopy structure of the human NaCT was solved in an inward-facing conformation. This was an important advancement in the NaCT field, paving the way for a better understanding of the structure-function relationships for this clinically important transporter. We classified 22 NaCT missense disease-causing mutations based on their localizations in the 3D structure. Class I mutations interfere with the transport function by blocking the elevator-type mechanism for substrate translocation. Class II cause defects in protein folding and protein trafficking to the cell surface, which may be corrected by small molecule therapeutics. As there are no NaCT-specific antibodies, we expressed WT and the mutants with specific epitopes to facilitate detection, which didn’t interfere with the presentation of the mutant phenotypes. The Class I mutations C50R, T142M, and T227M displayed protein and surface expression levels similar to WT. Class II mutants G219R, S427L, and L488P showed significantly decreased protein expression and no plasma membrane expression. Both classes displayed diminished transport activity. These experiments have brought us one step closer to understanding the defects of disease-causing mutations at the molecular level, allowing us to begin dissecting NaCT trafficking pathway(s).

Structural mechanism of SGLT1 inhibitors

Sodium glucose co-transporters (SGLT) harness the electrochemical gradient of sodium to drive the uphill transport of glucose across the plasma membrane. Human SGLT1 (hSGLT1) plays a key role in sugar uptake from food and its inhibitors show promise in the treatment of several diseases. However, the inhibition mechanism for hSGLT1 remains elusive. Here, we present the cryo-EM structure of the hSGLT1-MAP17 hetero-dimeric complex in the presence of the high-affinity inhibitor LX2761. LX2761 locks the transporter in an outward-open conformation by wedging inside the substrate-binding site and the extracellular vestibule of hSGLT1. LX2761 blocks the putative water permeation pathway of hSGLT1. The structure also uncovers the conformational changes of hSGLT1 during transitions from outward-open to inward-open states.

First-in-man trial of b3-adrenoreceptor agonist treatment in chronic heart failure – impact on diastolic function

Abstract Background Diastolic dysfunction (DD) in heart failure (HF) is associated with increased myocardial cytosolic calcium, and calcium-efflux via the sodium-calcium-exchanger depends on the sodium gradient. Beta-3-adrenoceptor (β3-AR)-agonist lowers cytosolic sodium and has been shown to reverse organ congestion. Purpose To assess whether β3-AR-agonist treatment improves DD. Methods In a first-in-man randomized controlled, double-blind trial, we assigned 70 patients with HF with reduced ejection fraction (HFrEF) (NYHA II–III) and LVEF &amp;lt;40% to receive mirabegron (300 mg/day) or placebo for 6 months, in addition to recommended HF-therapy. Patients were assessed with echocardiography and cardiac computed tomography (CCT) at baseline and follow-up. DD was graded according to the current American/European guidelines. Results Baseline and follow-up echocardiographic data were available in 57 patients (59±11 years, 88% male, 49% ischemic heart disease). Baseline LVEF was 34%±8%. No significant change in DD grade was found between the groups at follow-up, p=0.72. Neither was there any clinical differences in any singular diastolic parameters within or between groups by echocardiography (E/e' placebo: 13.3±6.9 to 12.6±5.1, p=0.19 vs. mirabegron: 12.0±5.7 to 12.8±7.9, p=0.67, mean difference 1.12 [95% CI −1.68 to 4.3], p=0.37), or CCT (left atrial max volume index: between group mean difference 0.2 [95% CI −6.2 to 5.6] ml/m2, p=0.91). Conclusions In patients with HFrEF, no improvement nor worsening in DD gradings or singular diastolic parameters after β3-AR stimulation compared to placebo were identified. The findings add to previous literature questioning the role of impaired Na+-Ca2+ mediated Ca2+ export as a major culprit in DD. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): The Heart Centre Research Foundation, RigshospitaletThe Novo Nordic Foundation

Autophagy-dependent Na+-K+-ATPase signalling and abnormal urate reabsorption in hyperuricaemia-induced renal tubular injury

Increasing evidence indicates that hyperuricaemia (HUA) is not only a result of decreased renal urate excretion but also a contributor to kidney disease. Na+-K+-ATPase (NKA), which establishes the sodium gradient for urate transport in proximal tubular epithelial cells (PTECs), its impairment leads to HUA-induced nephropathy. However, the specific mechanism underlying NKA impairment-mediated renal tubular injury and increased urate reabsorption in HUA is not well understood. In this study, we investigated whether autophagy plays a key role in the NKA impairment signalling and increased urate reabsorption in HUA-induced renal tubular injury. Protein spectrum analysis of exosomes from the urine of HUA patients revealed the activation of lysosomal processes, and exosomal expression of lysosomal-associated membrane protein-2 was associated with increased serum levels and decreased renal urate excretion in patients. We demonstrated that high uric acid (UA) induced lysosome dysfunction, autophagy and inflammation in a time- and dose-dependent manner and that high UA and/or NKA α1 siRNA significantly increased mitochondrial abnormalities, such as reductions in mitochondrial respiratory complexes and cellular ATP levels, accompanied by increased apoptosis in cultured PTECs. The autophagy inhibitor hydroxychloroquine (HCQ) ameliorated NKA impairment-mediated mitochondrial dysfunction, Nod-like receptor pyrin domain-containing protein 3 (NLRP3)-interleukin-1β (IL-1β) production, and abnormal urate reabsorption in PTECs stimulated with high UA and in rats with oxonic acid (OA)-induced HUA. Our findings suggest that autophagy plays a pivotal role in NKA impairment-mediated signalling and abnormal urate reabsorption in HUA-induced renal tubular injury and that inhibition of autophagy by HCQ could be a promising treatment for HUA.