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Funder: Dutch Kidney Foundation, AIMS: A potassium replete diet is associated with lower blood pressure (BP) and lower risk of cardiovascular disease (CVD). Whether these associations differ between men and women and whether they depend on daily sodium intake is unknown. METHODS AND RESULTS: An analysis was performed in 11 267 men and 13 696 women from the EPIC-Norfolk cohort. Twenty-four hour excretion of sodium and potassium, reflecting intake, was estimated from sodium and potassium concentration in spot urine samples using the Kawasaki formula. Linear and Cox regression were used to explore the association between potassium intake, systolic BP (SBP), and CVD events (defined as hospitalization or death due to CVD). After adjustment for confounders, interaction by sex was found for the association between potassium intake and SBP (P < 0.001). In women, but not in men, the inverse slope between potassium intake and SBP was steeper in those within the highest tertile of sodium intake compared with those within the lowest tertile of sodium intake (P < 0.001 for interaction by sodium intake). Both in men and women, higher potassium intake was associated with a lower risk of CVD events, but the hazard ratio (HR) associated with higher potassium intake was lower in women than in men [highest vs. lowest potassium intake tertile: men: HR 0.93, 95% confidence interval (CI) 0.87-1.00; women: HR 0.89, 95% CI 0.83-0.95, P = 0.033 for interaction by sex]. CONCLUSION: The association between potassium intake, SBP, and CVD events is sex specific. The data suggest that women with a high sodium intake in particular benefit most from a higher potassium intake with regard to SBP.

We compared the regulation of the NaCl cotransporter (NCC) in adaptation to a low-K (LK) diet in male and female mice. We measured hydrochlorothiazide (HCTZ)-induced changes in urine volume (UV), glomerular filtration rate (GFR), absolute (ENa, EK), and fractional (FENa, FEK) excretion in male and female mice on control-K (CK, 1% KCl) and LK (0.1% KCl) diets for 7 days. With CK, NCC-dependent ENa and FENa were larger in females than males as observed previously. However, with LK, HCTZ-induced ENa and FENa increased in males but not in females, abolishing the sex differences in NCC function as observed in CK group. Despite large diuretic and natriuretic responses to HCTZ, EK was only slightly increased in response to the drug when animals were on LK. This suggests that the K-secretory apparatus in the distal nephron is strongly suppressed under these conditions. We also examined LK-induced changes in Na transport protein expression by Western blotting. Under CK conditions females expressed more NCC protein, as previously reported. LK doubled both total (tNCC) and phosphorylated NCC (pNCC) abundance in males but had more modest effects in females. The larger effect in males abolished the sex-dependence of NCC expression, consistent with the measurements of function by renal clearance. LK intake did not change NHE3, NHE2, or NKCC2 expression, but reduced the amount of the cleaved (presumably active) form of γENaC. LK reduced plasma K to lower levels in females than males. These results indicated that males had a stronger NCC-mediated adaptation to LK intake than females.

Therapeutics that inhibit enzymes, receptors, ion channels, and cotransporters have long been the mainstay of cardiovascular medicine. Now, oligonucleotide therapeutics offer a modern variation on this paradigm of protein inhibition. Rather than target a protein, however, small interfering ribonucleic acids and antisense oligonucleotides target the messenger RNA (mRNA) from which a protein is translated. Endogenous, cellular mechanisms enable the oligonucleotides to bind a selected sequence on a target mRNA, leading to its degradation. The catalytic nature of the process confers an advantage over the stoichiometric binding of traditional small molecule therapeutics to their respective protein targets. Advances in nucleic acid chemistry and delivery have enabled development of oligonucleotide therapeutics against a wide range of diseases, including hyperlipidemias and hereditary transthyretin-mediated amyloidosis with polyneuropathy. While most of these therapeutics were initially designed for rare diseases, recent clinical trials highlight the potential impact of oligonucleotides on more common forms of cardiovascular disease.

Solid organ transplantation offers life-saving treatment for patients with end-organ dysfunction. Patient survival and quality of life have improved over the past few decades as a result of pharmacological development, expansion of the donor pool, technological advances and standardization of practices related to transplantation. Still, transplantation is associated with cardiovascular complications, of which post-transplant diabetes mellitus (PTDM) is one of the most important. PTDM increases mortality, which is best documented in patients who have received kidney and heart transplants. PTDM results from traditional risk factors seen in patients with type 2 diabetes mellitus, but also from specific post-transplant risk factors such as metabolic side effects of immunosuppressive drugs, post-transplant viral infections and hypomagnesemia. Oral hypoglycaemic agents are the first choice for the treatment of type 2 diabetes mellitus in non-transplanted patients. However, the evidence on the safety and efficacy of oral hypoglycaemic agents in transplant recipients is limited. The favourable risk/benefit ratio, which is suggested by large-scale and long-term studies on new glucose-lowering drug classes such as glucagon-like peptide 1 receptor agonists and sodium-glucose cotransporter 2 inhibitors, makes studies warranted to assess the potential role of these agents in the management of PTDM.

Supplemental Digital Content is available in the text., Background: SGLT2 (sodium/glucose cotransporter 2) inhibitors exert robust cardioprotective effects against heart failure in patients with diabetes, and there is intense interest to identify the underlying molecular mechanisms that afford this protection. Because the induction of the late component of the cardiac sodium channel current (late-INa) is involved in the etiology of heart failure, we investigated whether these drugs inhibit late-INa. Methods: Electrophysiological, in silico molecular docking, molecular, calcium imaging, and whole heart perfusion techniques were used to address this question. Results: The SGLT2 inhibitor empagliflozin reduced late-INa in cardiomyocytes from mice with heart failure and in cardiac Nav1.5 sodium channels containing the long QT syndrome 3 mutations R1623Q or ΔKPQ. Empagliflozin, dapagliflozin, and canagliflozin are all potent and selective inhibitors of H2O2-induced late-INa (half maximal inhibitory concentration = 0.79, 0.58, and 1.26 µM, respectively) with little effect on peak sodium current. In mouse cardiomyocytes, empagliflozin reduced the incidence of spontaneous calcium transients induced by the late-INa activator veratridine in a similar manner to tetrodotoxin, ranolazine, and lidocaine. The putative binding sites for empagliflozin within Nav1.5 were investigated by simulations of empagliflozin docking to a three-dimensional homology model of human Nav1.5 and point mutagenic approaches. Our results indicate that empagliflozin binds to Nav1.5 in the same region as local anesthetics and ranolazine. In an acute model of myocardial injury, perfusion of isolated mouse hearts with empagliflozin or tetrodotoxin prevented activation of the cardiac NLRP3 (nuclear-binding domain-like receptor 3) inflammasome and improved functional recovery after ischemia. Conclusions: Our results provide evidence that late-INa may be an important molecular target in the heart for the SGLT2 inhibitors, contributing to their unexpected cardioprotective effects.

Intestinal organoids derived from single cells undergo complex crypt–villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Here, using light-sheet microscopy and large-scale imaging quantification, we demonstrate that crypt formation coincides with a stark reduction in lumen volume. We develop a 3D biophysical model to computationally screen different mechanical scenarios of crypt morphogenesis. Combining this with live-imaging data and multiple mechanical perturbations, we show that actomyosin-driven crypt apical contraction and villus basal tension work synergistically with lumen volume reduction to drive crypt morphogenesis, and demonstrate the existence of a critical point in differential tensions above which crypt morphology becomes robust to volume changes. Finally, we identified a sodium/glucose cotransporter that is specific to differentiated enterocytes that modulates lumen volume reduction through cell swelling in the villus region. Together, our study uncovers the cellular basis of how cell fate modulates osmotic and actomyosin forces to coordinate robust morphogenesis. Yang, Xue et al. demonstrate in intestinal organoids that region-specific cell fates drive actomyosin patterns and modulate luminal osmotic forces to coordinate morphogenesis.

Background Sodium lactate (SL) has been described as an efficient therapy in treating raised intracranial pressure (ICP). However, the precise mechanism by which SL reduces intracranial hypertension is not well defined. An antiedematous effect has been proposed but never demonstrated. In this context, the involvement of chloride channels, aquaporins, or K–Cl cotransporters has also been suggested, but these mechanisms have never been assessed when using SL. Methods In a rat model of traumatic brain injury (TBI), we compared the effect of SL versus mannitol 20% on ICP, cerebral tissue oxygen pressure, and brain water content. We attempted to clarify the involvement of chloride channels in the antiedematous effects associated with lactate therapy in TBI. Results An equimolar single bolus of SL and mannitol significantly reduced brain water content and ICP and improved cerebral tissue oxygen pressure 4 h after severe TBI. The effect of SL on brain water content was much longer than that of mannitol and persisted at 24 h post TBI. Western blot and immunofluorescence staining analyses performed 24 h after TBI revealed that SL infusion is associated with an upregulation of aquaporin 4 and K–Cl cotransporter 2. Conclusions SL is an effective therapy for treating brain edema after TBI. This study suggests, for the first time, the potential role of chloride channels in the antiedematous effect induced by exogenous SL.

Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl− (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.

The epithelial Na+-coupled phosphate cotransporter family Slc34a (NaPi-II) is well conserved in vertebrates and plays an essential role in maintaining whole body levels of inorganic phosphate (Pi). A three-dimensional model of the transport protein has recently been proposed with defined substrate coordination sites. Zebrafish express two NaPi-II isoforms with high sequence identity but a 10-fold different apparent Km for Pi ([Formula: see text]). We took advantage of the two zebrafish isoforms to investigate the contribution of specific amino acids to Pi coordination and transport. Mutations were introduced to gradually transform the low-affinity isoform into a high-affinity transporter. The constructs were expressed in Xenopus laevis oocytes and functionally characterized. Becaue the cotransport of Pi and Na involves multiple steps that could all influence [Formula: see text], we performed a detailed functional analysis to characterize the impact of the mutations on particular steps of the transport cycle. We used varying concentrations of the substrates Pi and its slightly larger analog, arsenate, as well as the cosubstrate, Na+. Moreover, electrogenic kinetics were performed to assess intramolecular movements of the transporter. All of the mutations were found to affect multiple transport steps, which suggested that the altered amino acids induced subtle structural changes rather than coordinating Pi directly. The likely positions of the critical residues were mapped to the model of human Slc34a, and their localization in relation to the proposed substrate binding pockets concurs well with the observed functional data.

Key points The electrogenic Na+/HCO3 −cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT).The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies.Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B.We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain. Abstract The electrogenic Na+/HCO3 − cotransporter NBCe1‐B is widely expressed in many tissues in the body. NBCe1‐B exhibits only basal activity due to the action of the auto‐inhibition domain (AID) in its unique amino‐terminus. However, NBCe1‐B can be activated by interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto‐inhibition of NBCe1‐B and its activation by IRBIT. The IRBIT‐binding domain (IBD) of NBCe1‐B spans residues 1−52, essentially consisting of two arms, one negatively charged (residues 1−24) and the other positively charged (residues 40−52). The AID mainly spans residues 40−85, overlapping with the IBD in the positively charged arm. The magnitude of auto‐inhibition of NBCe1‐B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1‐B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1‐B; (2) IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain., Key points The electrogenic Na+/HCO3 −cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT).The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies.Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B.We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain.

The renal distal convoluted tubule (DCT) is critical for the fine-tuning of urinary ion excretion and the control of blood pressure. Ion transport along the DCT is tightly controlled by posttranscriptional mechanisms including a complex interplay of kinases, phosphatases, and ubiquitin ligases. Previous work identified the transcription factor Prox-1 as a gene significantly enriched in the DCT of adult mice. To test if Prox-1 contributes to the transcriptional regulation of DCT function and structure, we developed a novel mouse model (NCCcre:Prox-1flox/flox) for an inducible deletion of Prox-1 specifically in the DCT. The deletion of Prox-1 had no obvious impact on DCT structure and growth independent whether the deletion was achieved in newborn or adult mice. Furthermore, DCT-specific Prox-1 deficiency did not alter DCT-proliferation in response to loop diuretic treatment. Likewise, the DCT-specific deletion of Prox-1 did not cause other gross phenotypic abnormalities. Body weight, urinary volume, Na+ and K+ excretion as well as plasma Na+, K+, and aldosterone levels were similar in Prox-1DCTKO and Prox-1DCTCtrl mice. However, Prox-1DCTKO mice exhibited a significant hypomagnesemia with a profound downregulation of the DCT-specific apical Mg2+ channel TRPM6 and the NaCl cotransporter (NCC) at both mRNA and protein levels. The expression of other proteins involved in distal tubule Mg2+ and Na+ handling was not affected. Thus, Prox-1 is a DCT-enriched transcription factor that does not control DCT growth but contributes to the molecular control of DCT-dependent Mg2+ homeostasis in the adult kidney.

Aims Emipagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose co-transporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na+/H+ exchanger 1 (NHE1), causing a reduction in intracellular [Na+] ([Na+]i). However, at resting intracellular pH (pHi), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na+]i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. Methods and results The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pHi recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10, or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na+]i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na+]i rise upon superfusion of Na+-depleted cells with Na+-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na+]i at baseline nor pHi recovery following acute acidosis, as measured by 23Na triple quantum filtered NMR and 31P NMR, respectively. Conclusions Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na+]i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na+]., Graphical Abstract

Chloride homeostasis, the main determinant factor for the dynamic tuning of GABAergic inhibition during development, has emerged as a key element altered in a wide variety of brain disorders. Accordingly, developmental disorders such as schizophrenia, Autism Spectrum Disorder, Down syndrome, epilepsy, and tuberous sclerosis complex (TSC) have been associated with alterations in the expression of genes codifying for either of the two cotransporters involved in the excitatory-to-inhibitory GABA switch, KCC2 and NKCC1. These alterations can result from environmental insults, including prenatal stress and maternal separation which share, as common molecular denominator, the elevation of pro-inflammatory cytokines. In this review we report and systemize recent research articles indicating that different perinatal environmental perturbations affect the expression of chloride transporters, delaying the developmental switch of GABA signaling, and that inflammatory cytokines, in particular interleukin 1β, may represent a key causal factor for this phenomenon. Based on literature data, we provide therefore a unifying conceptual framework, linking environmental hits with the excitatory-to-inhibitory GABA switch in the context of brain developmental disorders.

Na+–K+–Cl− cotransporter 2 (NKCC2; SLC12A1) is an integral membrane protein that comes as three splice variants and mediates the cotranslocation of Na+, K+, and Cl− ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, and through the involvement of other ion transport systems, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na+, Cl−, Ca2+, Mg2 +, and HCO3 − loads. The functional relevance of NKCC2 in human is illustrated by the many abnormalities that result from the inactivation of this transport system through the use of loop diuretics or in the setting of inherited disorders. The following presentation aims at discussing the physiological roles and molecular characteristics of Na+–K+–Cl− cotransport in the TALH and those of the individual NKCC2 splice variants more specifically. Many of the historical and recent data that have emerged from the experiments conducted will be outlined and their larger meaning will also be placed into perspective with the aid of various hypotheses., Na+–K+–Cl− cotransporter 2 (NKCC2) mediates the cotranslocation of Na+, K+, and Cl− ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na+, Cl−, Ca2+, Mg2 +, and HCO3 − load. The functional relevance of NKCC2 in humans is illustrated by the many abnormalities that result from the inactivation of this transport system.

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+–2HCO3− stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3− influx. However, NBCe1 in the kidney mediates HCO3− efflux (HCO3− reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3− efflux) than in murine colons (in which NBCe1 mediates HCO3− influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.

Inhibition of the sodium-glucose cotransporter 2 (SGLT2) by canagliflozin in type 2 diabetes mellitus results in large between-patient variability in clinical response. To better understand this variability, the positron emission tomography (PET) tracer [18F]canagliflozin was developed via a Cu-mediated 18F-fluorination of its boronic ester precursor with a radiochemical yield of 2.0 ± 1.9% and a purity of >95%. The GMP automated synthesis originated [18F]canagliflozin with a yield of 0.5-3% (n = 4) and a purity of >95%. Autoradiography showed [18F]canagliflozin binding in human kidney sections containing SGLT2. Since [18F]canagliflozin is the isotopologue of the extensively characterized drug canagliflozin and thus shares its toxicological and pharmacological characteristics, it enables its immediate use in patients.

Background: Recent clinical studies demonstrate that SGLT2 (sodium-glucose cotransporter 2) inhibitors ameliorate heart failure (HF). The present study was conducted to assess the expression and function of renal SGLT2 and the influence of enhanced renal sympathetic tone in HF. Methods: Four weeks after coronary artery ligation surgery to induce HF, surgical bilateral renal denervation (RDN) was performed in rats. Four groups of rats (Sham-operated control [Sham], Sham+RDN, HF and HF+RDN; n=6/group) were used. Immunohistochemistry and Western blot analysis were performed to evaluate the renal SGLT2 expression. One week after RDN (5 weeks after induction of HF), intravenous injection of SGLT2 inhibitor dapagliflozin were performed to assess renal excretory responses. In vitro, human embryonic kidney cells were used to investigate the fractionation of SGLT2 after norepinephrine treatment. Results: In rats with HF, (1) SGLT2 expression in the proximal tubule of the kidney was increased; (2) the response of increases in urine flow, sodium excretion, and glucose excretion to dapagliflozin were greater; and (3) RDN attenuated renal SGLT2 expression and normalized renal functional responses to dapagliflozin. In vitro, norepinephrine promoted translocation of SGLT2 to the cell membrane. Conclusions: These results indicate that the enhanced tonic renal sympathetic nerve activation in HF increases the expression and functional activity of renal SGLT2. Potentiated trafficking of SGLT2 to cell surface in renal proximal tubules mediated by norepinephrine may contribute to this functional activation of SGLT2 in HF. These findings provide critical insight into the underlying mechanisms for the beneficial effects of SGLT2 inhibitors on HF reported in the clinical studies.

Cation-coupled chloride cotransporters play a key role in generating the Cl– electrochemical gradient on the cell membrane, which is important for regulation of many cellular processes. However, a quantitative analysis of the interplay between numerous membrane transporters and channels in maintaining cell ionic homeostasis is still undeveloped. Here, we demonstrate a recently developed approach on how to predict cell ionic homeostasis dynamics when stopping the sodium pump in human lymphoid cells U937. The results demonstrate the reliability of the approach and provide the first quantitative description of unidirectional monovalent ion fluxes through the plasma membrane of an animal cell, considering all the main types of cation-coupled chloride cotransporters operating in a system with the sodium pump and electroconductive K+, Na+, and Cl– channels. The same approach was used to study ionic and water balance changes associated with regulatory volume decrease (RVD), a well-known cellular response underlying the adaptation of animal cells to a hypoosmolar environment. A computational analysis of cell as an electrochemical system demonstrates that RVD may happen without any changes in the properties of membrane transporters and channels due to time-dependent changes in electrochemical ion gradients. The proposed approach is applicable when studying truly active regulatory processes mediated by the intracellular signaling network. The developed software can be useful for calculation of the balance of the unidirectional fluxes of monovalent ions across the cell membrane of various cells under various conditions.

Angiotensin receptor blockers (ARBs) and sodium-glucose cotransporter 2 inhibitors (SGLT2i) have been used as the standard therapy for patients with diabetic kidney disease (DKD). However, how these two drugs possess additive renoprotective effects remains unclear. Here, we conducted single-cell RNA sequencing to profile the kidney cell transcriptome of db/db mice treated with vehicle, ARBs, SGLT2i, or ARBs plus SGLT2i, using db/m mice as control. We identified 10 distinct clusters of kidney cells with predominant proximal tubular (PT) cells. We found that ARBs had more anti-inflammatory and anti-fibrotic effects, while SGLT2i affected more mitochondrial function in PT. We also identified a new PT subcluster, was increased in DKD, but reversed by the treatments. This new subcluster was also confirmed by immunostaining of mouse and human kidneys with DKD. Together, our study reveals kidney cell-specific gene signatures in response to ARBs and SGLT2i and identifies a new PT subcluster, which provides new insight into the pathogenesis of DKD.

Aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a core circadian transcription factor that controls the 24-h cycle of physiological processes. In shrimp, the role of BMAL1 in the regulating glucose metabolism remains unclear. Firstly, we observed that the daily profile of BMAL1, GLUT1 and SGLT1 expression were synchronized in the intestine and the hepatopancreas of Litopenaeus vannamei. Then we examined the effects of BMAL1 on the gene expression of glucose transporter type 1 (SGLT1) and sodium-glucose cotransporter 1 (GLUT1) in vivo and in vitro. BMAL1 in L. vannamei shares 70.91–96.35% of sequence identities with other shrimp species and possesses the conserved helix-loop-helix domain and polyadenylation site domain. The in vitro dual-luciferase reporter assay and in vivo RNA interference experiment demonstrated that BMAL1 exerted a positive regulation effect on the expression of glucose transporters in L. vannamei. Moreover, we conducted an eight-week treatment to investigate whether light/dark cycle change would influence growth performance, and gene expression of BMAL1, GLUT1 and SGLT1 in L. vannamei. Our result showed that compared with natural light treatment, constant darkness (24-hour darkness) significantly decreased (P &lt, 0.05) serum glucose concentration, and suppressed (p &lt, 0.05) the gene expression of BMAL1, GLUT1 and SGLT1 in the hepatopancreas and the intestine. Growth performance and survival rate were also decreased (p &lt, 0.05) by constant darkness treatment. Our result identified BMAL1 as a critical mediator regulating the expression of glucose transporters, which could be suppressed by constant darkness in L. vannamei. It would be quite interesting to explore the mechanism of dark/light cycles on glucose transport and metabolism in L. vannamei, which might provide a feeding strategy for improving carbohydrate utilization in the future.

First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel antiseizure strategies. Impaired GABAergic inhibition is key to the development of such benzodiazepine-resistant seizures, as well as the pathophysiology of status epilepticus (SE). It is emerging that reduced or impaired neuronal K+/Cl- cotransporter 2 (KCC2) activity contributes to deficits in γ-aminobutyric acid (GABA)-mediated inhibition and increased seizure vulnerability. The with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signaling pathway inhibits neuronal KCC2 via KCC2-T1007 phosphorylation. A selective WNK kinase inhibitor, WNK463, was recently synthesized by Novartis. Exploiting WNK463, we test the hypothesis that pharmacological WNK inhibition will enhance KCC2 activity, increase the efficacy of GABAergic inhibition, and thereby limit seizure activity in animal models. Immunoprecipitation and Western blot analysis were used to examine WNK463's effects on KCC2-T1007 phosphorylation, in vitro and in vivo. A thallium (Tl+) uptake assay was used in human embryonic kidney (HEK-293) cells expressing KCC2 to test WNK463's effects on KCC2-mediated Tl+ transport. Gramicidin-perforated- and whole-cell patch-clamp recordings in cortical rat neurons were used to examine WNK463's effects on KCC2-mediated Cl- transport. In mouse brain slices (entorhinal cortex), field recordings were utilized to examine WNK463's effects on 4-aminopyridine-induced seizure activity. Last, WNK463 was directly deliver to the mouse hippocampus in vivo and tested in a kainic acid model of diazepam-resistant SE. WNK463 significantly reduces KCC2-T1007 phosphorylation in vitro and in vivo (mice). In human embryonic kidney 293 (HEK-293) cells expressing KCC2, WNK463 greatly enhanced the rates Tl+ transport. However, the drug did not enhance Tl+ transport in cells expressing a KCC2-phospho null T1007 mutant. In cultured rat neurons, WNK463 rapidly reduced intracellular Cl- and consequently hyperpolarized the Cl- reversal potential (EGABA). In mature neurons that were artificially loaded with 30 mM Cl-, WNK463 significantly enhanced KCC2-mediated Cl- export and hyperpolarized EGABA. In a 4-aminopyridine model of acute seizures, WNK463 reduced the frequency and number of seizure-like events (SLEs). Finally, in an in vivo kainic acid (KA) model of diazepam-resistant SE, WNK463 slowed the onset and reduced the severity of KA-induced status epilepticus. Last, WNK463 prevented the development of pharmaco-resistance to diazepam in drug-treated mice. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. Our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant SE.

Rapid postprandial blood glucose elevation can cause lifestyle-related diseases, such as type II diabetes. The absorption of food-derived glucose is primarily mediated by sodium/glucose cotransporter 1 (SGLT1). Moderate SGLT1 inhibition can help attenuate postprandial blood glucose elevation and prevent lifestyle-related diseases. In this study, we established a CHO cell line stably expressing human SGLT1 and examined the effects of phytochemicals on SGLT1 activity. Among the 50 phytochemicals assessed, tangeretin and cardamonin inhibited SGLT1 activity. Tangeretin and cardamonin did not affect the uptake of L-leucine, L-glutamate, and glycyl-sarcosine. Tangeretin, but not cardamonin, inhibited fructose uptake, suggesting that the inhibitory effect of tangeretin was specific to the monosaccharide transporter, whereas that of cardamonin was specific to SGLT1. Kinetic analysis suggested that the suppression of SGLT1 activity by tangeretin was associated with a reduction in Vmax and an increase in Km, whereas suppression by cardamonin was associated with a reduction in Vmax and no change in Km. Oral glucose tolerance tests in mice showed that tangeretin and cardamonin significantly suppressed the rapid increase in blood glucose levels. In conclusion, tangeretin and cardamonin were shown to inhibit SGLT1 activity in vitro and lower blood glucose level in vivo.

Aim: The phosphorylation level of the furosemide-sensitive Na +-K +-2Cl − cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti-pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform-specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice. Methods: Genetic information, molecular biology, biochemical techniques and mouse phenotyping was used to study NKCC2 and kidney function in two commonly used mouse strains (ie 129Sv and in C57BL/6 mice). Moreover, a new phosphoform-specific mouse NKCC2 antibody was developed and characterized. Results: Amino acids sequence alignment revealed that C57BL/6 mice have a strain-specific five amino acids deletion (ΔF97-T101) in NKCC2 that diminishes the detection of NKCC2 phosphorylation with previously developed pT96/pT101 NKCC2 antibodies. Instead, the antibodies cross-react with the phosphorylated thiazide-sensitive NaCl cotransporter (NCC), which can obscure interpretation of results. Interestingly, the deletion in NKCC2 does not impact on kidney function and/or expression of renal ion transport proteins as indicated by the analysis of the F2 generation of crossbred 129Sv and C57BL/6 mice. A newly developed pT96 NKCC2 antibody detects pNKCC2 in both mouse strains and shows no cross-reactivity with phosphorylated NCC. Conclusion: Our work reveals a hitherto unappreciated, but essential, strain difference in the amino acids sequence of mouse NKCC2 that needs to be considered when analysing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat.

Background: Type 2 diabetes mellitus (T2DM) is an independent risk factor for cardiovascular impairment, increasing the rates of atherosclerotic and non-atherosclerotic events. Additionally, adverse kidney events are directly linked with T2DM and cardiovascular diseases. In this context, the sodium-glucose cotransporter 2 inhibitors (SGLT2i) have demonstrated both cardioprotective and renoprotective effects in patients with or without T2DM. Therefore, the present meta-analysis aims to evaluate cardiovascular outcomes involving SGLT2i as monotherapy or other add-on antidiabetic agents (ADA) in patients with or without T2DM. Objetive: The present meta-analysis aims to evaluate cardiovascular outcomes involving SGLT2i as monotherapy or add-on other ADA in patients with or without T2DM. Methods: The entrance criteria to SGLT2i studies were: describing any data regarding cardiovascular effects; enrolling more than 1,000 participants; being approved by either the FDA or the EU, and having available access to the supplementary data. The trial had to exhibit at least one of the following results: major adverse cardiovascular events (MACE), cardiovascular death or hospitalization for heart failure, cardiovascular death, hospitalization for heart failure, renal or cardiovascular adverse events, or non-cardiovascular death. The significance level of 0.05 was adopted in the statistical analysis. Results: Nine trials with a total of 76,285 participants were included in the meta-analysis. SGLT2i reduced MACE (RR 0.75, 95% CI [0.55-1.01]), cardiovascular death or hospitalization for heart failure (RR 0.72, 95% CI [0.55-0.93]), cardiovascular death (RR 0.66, 95% CI [0.48-0.91]), hospitalization for heart failure (RR 0.58, 95% CI [0.46-0.73]), renal or cardiovascular adverse events (RR 0.55, 95% CI [0.39-0.78]), and non-cardiovascular death (RR 0.88, 95% CI [0.60-1.00]). Conclusions: Conjunction overall data suggests that these drugs can minimize the risk of cardiovascular events, thus decreasing mortality in patients, regardless of the presence of T2DM.

Glucose-galactose malabsorption (GGM) is due to mutations in the gene coding for the intestinal sodium glucose cotransporter SGLT1 (SLC5A1). Here we identify the rare variant Gln457Arg (Q457R) in a large pedigree of patients in the Västerbotten County in Northern Sweden with the clinical phenotype of GGM. The functional effect of the Q457R mutation was determined in protein expressed in Xenopus laevis oocytes using biophysical and biochemical methods. The mutant failed to transport the specific SGLT1 sugar analog α-methyl-D-glucopyranoside (αMDG). Q457R SGLT1 was synthesized in amounts comparable to the wild-type (WT) transporter. SGLT1 charge measurements and freeze-fracture electron microscopy demonstrated that the mutant protein was inserted into the plasma membrane. Electrophysiological experiments, both steady-state and presteady-state, demonstrated that the mutant bound sugar with an affinity lower than the WT transporter. Together with our previous studies on Q457C and Q457E mutants, we established that the positive charge on Q457R prevented the translocation of sugar from the outward-facing to inward-facing conformation. This is contrary to other GGM cases where missense mutations caused defects in trafficking SGLT1 to the plasma membrane. Thirteen GGM patients are now added to the pedigree traced back to the late 17th century. The frequency of the Q457R variant in Västerbotten County genomes, 0.0067, is higher than in the general Swedish population, 0.0015, and higher than the general European population, 0.000067. This explains the high number of GGM cases in this region of Sweden.

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24-48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP.

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT.

Salbutamol activates the NaCl cotransporter of the distal convoluted tubule. Salbutamol, in conjunction with high salt intake, induced hypertension in mice, rescued by thiazide therapy. Phosphoproteomics identified protein phosphatase 1/inhibitor 1 as a distinct regulatory node for NaCl cotransporter activation by salbutamol, which did not activate the transporter in inhibitor 1 knockout mice. Salbutamol is widely used in respiratory medicine, and the acquisition of salt sensitivity may be relevant to understanding cardiovascular risk in certain patients.

Administration of an sodium-glucose cotransporter 2 inhibitor to individuals with insulin resistance reduces the level of glucose disposal necessary to maintain glycemia as a result of the induced increase in urinary glucose excretion. The amount of insulin required for a certain level of glucose disposal, on which the evaluation of insulin resistance is usually based, might thus decrease even without amelioration of insulin resistance.

Type A insulin resistance (IR) syndrome is a severe IR form caused by insulin receptor (INSR) gene defects. Antidiabetic drugs, including high‐dose insulin and insulin‐sensitizing agents, often fail to control associated hyperglycemia. Therapy with recombinant human insulin‐like growth factor 1 can be more effective, but it is expensive. We report a case of type A IR syndrome with an in‐frame INSR heterozygous deletion (ΔLeu999) that was treated with a combination of conventional therapy and ipragliflozin, a sodium–glucose cotransporter 2 inhibitor. Treatment reduced hemoglobin A1c levels (10.0–7.5%) and induced weight loss (54.4–52.0 kg) within 2 months, and the effects were sustained for >3 years. Sodium–glucose cotransporter 2 inhibitors might be useful to normalize blood glucose in type A IR syndrome by reducing bodyweight and ameliorating glucotoxicity., This is the first article to report on the long‐term efficacy of ipragliflozin (a sodium–glucose cotransporter 2 inhibitor) in a patient with insulin receptor mutation. Our findings can be helpful in the development of more effective and affordable treatment strategies for patients with severe insulin resistance syndrome.

The SLC12A cation-Cl− cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (“ZT-1a”). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.

The effect of sodium‐glucose cotransporter 2 inhibitor (SGLT2I) on nonalcoholic steatohepatitis (NASH) has been reported, but there are few studies on its effect on NASH‐related renal injury. In this study, we examined the effect of SGLT2I using a novel medaka fish model of NASH‐related kidney disease, which was developed by feeding the d‐rR/Tokyo strain a high‐fat diet. SGLT2I was administered by dissolving it in water of the feeding tank. SGLT2I ameliorates macrophage accumulation and oxidative stress and maintained mitochondrial function in the kidney. The results demonstrate the effect of SGLT2I on NASH‐related renal injury and the usefulness of this novel animal model for research into NASH‐related complications., Here, we demonstrate that the highly specific sodium‐glucose cotransporter 2 inhibitor (SGLT2I) prevented the progression of nonalcoholic steatohepatitis (NASH)‐related renal injury in a medaka fish model of NASH‐related kidney disease. SGLT2I ameliorates oxidative stress and macrophage accumulation and maintained mitochondrial function in renal tubules.

The With-No-Lysine (WNK) kinase is considered to be a master regulator for various cation-chloride cotransporters involved in maintaining cell-volume and ion homeostasis. Here, we have investigated the phosphorylation-induced structural dynamics of the WNK1 kinase bound to an inhibitor via atomistic molecular dynamics simulations. Results from our simulations show that the phosphorylation at Ser382 could stabilize the otherwise flexible activation loop (A-loop). The intrahelix salt-bridge formed between Arg264 and Glu268 in the unphosphorylated system is disengaged after the phosphorylation, and Glu268 reorients itself and forms a stable salt-bridge with Arg348. The dynamic cross-correlation analysis shows that phosphorylation diminishes anticorrelated motions and increases correlated motions between different domains. Structural network analysis reveals that the phosphorylation causes structural rearrangements and shortens the communication path between the αC-helix and catalytic loop, making the binding pocket more suitable for accommodating the ligand. Overall, we have characterized the structural changes in the WNK kinase because of phosphorylation in the A-loop, which might help in designing rational drugs.

It is widely known that intestinal capacities such as the enzymatic hydrolysis of carbohydrates, lipids and proteins, and the subsequent absorption of the hydrolyzed products, are evolutionary matched to dietary loads and feeding behaviors. In this study, we demonstrate that the protein expression of apically located sodium-dependent glucose cotransporter-1 (SGLT-1) throughout rat ontogeny is daily adjusted to afford glucose uptake when the load of this metabolically essential monosaccharide in the intestinal lumen is maximum. The jejunal expression of SGLT-1 protein in 14 one-day-old suckling pups was found to increase at dark and early light phase (P < 0.05), when they have a better access to mother milk. In weaning 21-d-old and juvenile 28-d-old rats, the cotransporter expression was high throughout the entire day (P < 0.05). Finally, adult 90-d-old rats showed a well-developed circadian rhythm for SGLT-1 protein (P < 0.05), whose expression increased at late light and dark phase when the highest intestinal glucose load was achieved. To our knowledge, these results are the first reporting the daily profile of SGLT-1 expression during rat early developmental stage and may contribute to understand the biological significance of a well-established molecular capacity to deal with the crucial increase of glucose load in the diet during the weaning process. Fil: Baston, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Cid, Fabricio Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina Fil: Caviedes Vidal, Enrique Juan Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina Fil: Chediack, Juan Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina

PURPOSE OF REVIEW: Sodium glucose cotransporter 2 (SGLT2) inhibitors are relatively novel antidiabetic drugs that improve glycemic control and reduce cardiovascular outcomes as well as renal function decline. SGLT2 inhibitors act by inhibiting glucose reabsorption in the proximal tubule of the kidney. Emerging data suggest that these drugs may also influence bone and mineral metabolism. This review summarizes clinical trial data on bone and mineral outcomes, and discusses potential underlying mechanisms.RECENT FINDINGS: Three large randomized controlled trials documented cardiovascular and renal protective effects of SGLT2 inhibitors. Recent studies indicate that SGLT2 inhibitors influence renal phosphate reabsorption and calciuria. Although the CANVAS trial suggested an increased fracture risk associated with canagliflozin compared with placebo, the vast majority of trials and meta-analyses did not demonstrate an increased fracture risk associated with SGLT2 inhibitor use.SUMMARY: SGLT2 inhibitors have shown clinically relevant cardiovascular and renal protective effects. The long-term implications for bone health, in particular in the context of chronic kidney disease, are still incompletely understood and warrant further investigation.

For type 1 diabetes patients with inadequate glycemic control, one treatment option is to increase the insulin dose (scenario 1), which should not give rise to a "metabolic imbalance." A second option is additional treatment with a sodium-glucose cotransporter 2 inhibitor, which might lead to a "metabolic imbalance" (scenario 2). A reduction in insulin dose in addition to administration of a sodium-glucose cotransporter 2 inhibitor might further increase the "metabolic imbalance" (scenario 3).

Summary: mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na+/K+/2Cl− cotransporter NKCC1 (SLC12A2), a known regulator of cell volume, is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity, as well as activation of Akt and Erk, leading to activation of mTORC1 in cells, colonic organoids, and mouse colon. Moreover, NKCC1 depletion reduces intracellular Na+ concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1, therefore, suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly, by linking ion transport and cell volume regulation to mTORC1 function, NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation. : Demian et al. show that the Na/K/2Cl ion transporter NKCC1, a known regulator of cell volume, inhibits the Leu transporter LAT1 and the Akt/Erk pathways, thus inhibiting mTORC1 activation and providing a link between cell volume (size) and cell mass regulation. Keywords: Leu transporter, LAT1-4F2hc, SLC7A5-SLC3A2, SLC12A2, Gln transporter, mTORC1, cell volume, cell mass

Melanoma cells preserve intracellular pH (pHi) within a viable range despite an acidic ambient pH that typically falls below pH 7.0. The molecular mechanisms underlying this form of acidic preservation in melanoma remain poorly understood. Previous studies had demonstrated that proton transporters including the monocarboxylate transporter (MCT), the sodium hydrogen exchanger (NHE), and V-Type ATPase mediate acid extrusion to counter intracellular acidification in melanoma cells. In this report, the expression and function of the Sodium-Coupled Bicarbonate Transporter (NCBT) family of base loaders were further characterized in melanoma cell lines. NCBT family members were found to be expressed in three different melanoma cell lines – A375, MeWo, and HS695T – and included the electrogenic sodium-bicarbonate cotransporter isoforms 1 and 2 (NBCe1 and NBCe2), the electroneutral sodium-bicarbonate cotransporter (NBCn1), and the sodium-dependent chloride-bicarbonate exchanger (NDCBE). These transporters facilitated 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-dependent pHi recovery in melanoma cells, in response to intracellular acidification induced by ammonium chloride prepulse. Furthermore, the expression of NCBTs were upregulated via chronic exposure to extracellular acidification. Given the current research interest in the NCBTs as a molecular driver of tumourigenesis, characterising NCBT in melanoma provides impetus for developing novel therapeutic targets for melanoma treatment.

Recent studies have suggested that postprandial increases in insulin directly contribute to reduced urinary sodium excretion. An abundance of research supports the ability of insulin to augment epithelial sodium channel (ENaC) transport. This study hypothesized that ENaC contributes to the increase in renal sodium reabsorption following a meal. To test this, we used fasted or 4 hour postprandial Sprague Dawley rats to analyze ENaC expression and activity. We also assessed total expression of additional sodium transporters (Na+-Cl− cotransporter (NCC), Na+-K+-2Cl− cotransporter (NKCC2), and Na+-K+-ATPase (NKA)) and circulating hormones involved in the renin-angiotensin-aldosterone system (RAAS). We found that after carbohydrate stimulus, ENaC open probability increased in split-open isolated collecting duct tubules, while ENaC protein levels remained unchanged. This was supported by a lack of change in phosphorylated Nedd4-2, an E3 ubiquitin ligase protein which regulates the number of ENaCs at the plasma membrane. Additionally, we found no differences in total expression of NCC, NKCC2, or NKA in the postprandial rats. Lastly, there were no significant changes in RAAS signaling between the stimulated and fasted rats, suggesting that acute hyperinsulinemia increases ENaC activity independent of the RAAS signaling cascade. These results demonstrate that insulin regulation of ENaC is a potential mechanism to preserve sodium and volume loss following a meal, and that this regulation is distinct from classical ENaC regulation by RAAS.

Phosphorus is one of the essential mineral elements of animals that plays an important role in animal growth and development, bone formation, energy metabolism, nucleic acid synthesis, cell signal transduction, and blood acid–base balance. It has been established that the Type IIb sodium-dependent phosphate cotransporters (NaPi-IIb) protein is the major sodium-dependent phosphate (Pi) transporter, which plays an important role in Pi uptake across the apical membrane of epithelial cells in the small intestine. Previous studies have demonstrated that epidermal growth factor (EGF) is involved in regulating intestinal Pi absorption. Here we summarize the effects of EGF on active Pi transport of NaPi-IIb under different conditions. Under normal conditions, EGF inhibits the active transport of Pi by inhibiting the expression of NaPi-IIb, while, under intestinal injury condition, EGF promotes the active absorption of Pi through upregulating the expression of NaPi-IIb. This review provides a reference for information about EGF-regulatory functions in Pi absorption in the animal intestine.

Background: The utility of urinary extracellular vesicles (uEVs) to faithfully represent the changes of renal tubular protein expression remains unclear. We aimed to evaluate renal tubular sodium (Na+) or potassium (K+) associated transporters expression from uEVs and kidney tissues in patients with Gitelman syndrome (GS) caused by inactivating mutations in SLC12A3.Methods: uEVs were isolated by ultracentrifugation from 10 genetically-confirmed GS patients. Membrane transporters including Na+-hydrogen exchanger 3 (NHE3), Na+/K+/2Cl− cotransporter (NKCC2), NaCl cotransporter (NCC), phosphorylated NCC (p-NCC), epithelial Na+ channel β (ENaCβ), pendrin, renal outer medullary K1 channel (ROMK), and large-conductance, voltage-activated and Ca2+-sensitive K+ channel (Maxi-K) were examined by immunoblotting of uEVs and immunofluorescence of biopsied kidney tissues. Healthy and disease (bulimic patients) controls were also enrolled.Results: Characterization of uEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Compared with healthy controls, uEVs from GS patients showed NCC and p-NCC abundance were markedly attenuated but NHE3, ENaCβ, and pendrin abundance significantly increased. ROMK and Maxi-K abundance were also significantly accentuated. Immunofluorescence of the representative kidney tissues from GS patients also demonstrated the similar findings to uEVs. uEVs from bulimic patients showed an increased abundance of NCC and p-NCC as well as NHE3, NKCC2, ENaCβ, pendrin, ROMK and Maxi-K, akin to that in immunofluorescence of their kidney tissues.Conclusion: uEVs could be a non-invasive tool to diagnose and evaluate renal tubular transporter adaptation in patients with GS and may be applied to other renal tubular diseases.

Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na(+) and/or K(+) with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K(+) gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins is challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our recent publication ( Liu et al., 2019 ). The procedure may be adapted for functional and structural studies.

Phosphorylation of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) is altered rapidly in response to changes in extracellular K(+) concentration ([K(+)]). High extracellular [K(+)] is believed to activate specific phosphatases to dephosphorylate NCC, thereby reducing its activity. This process is defective in the human disease familial hyperkalemic hypertension, in which extracellular [K(+)] fails to dephosphorylate NCC, suggesting an interplay between NCC-activating and NCC-inactivating switches. Here, we explored the role of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and intracellular Cl(−) concentration in the rapid effects of extracellular K(+) on NCC phosphorylation. SPAK was found to be rapidly dephosphorylated in vitro in human embryonic kidney cells and ex vivo in kidney slices by high [K(+)]. Acute high-K(+) challenge resulted in DCT1-specific SPAK dephosphorylation in vivo and dissolution of SPAK puncta. In line with the postulate of interplay between activating and inactivating switches, we found that the “on” switch, represented by with no lysine kinase 4 (WNK4)-SPAK, must be turned off for rapid NCC dephosphorylation by high [K(+)]. Longer-term WNK-SPAK-mediated stimulation, however, altered the sensitivity of the system, as it attenuated rapid NCC dephosphorylation due to acute K(+) loading. Although blockade of protein phosphatase (PP)1 increased NCC phosphorylation at baseline, neither PP1 nor PP3, singly or in combination, was essential for NCC dephosphorylation. Overall, our data suggest that NCC phosphorylation is regulated by a dynamic equilibrium between activating kinases and inactivating phosphatases, with kinase inactivation playing a key role in the rapid NCC dephosphorylation by high extracellular K(+). NEW & NOTEWORTHY Although a great deal is known about mechanisms by which thiazide-sensitive NaCl cotransporter is phosphorylated and activated, much less is known about dephosphorylation. Here, we show that rapid dephosphorylation by high K(+) depends on the Cl(−) sensitivity of with no lysine kinase 4 and the rapid dephosphorylation of STE20/SPS1-related proline-alanine-rich protein kinase, primarily along the early distal convoluted tubule.