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Type-III sodium-dependent phosphate transporters 1 and 2 (PiT1 and PiT2, respectively) are proteins encoded by SLC20A1 and SLC20A2, respectively. The ubiquitous distribution of SLC20A1 and SLC20A2 mRNAs in mammalian tissues supports the housekeeping maintenance and homeostasis of intracellular inorganic phosphate (Pi), which is absorbed from interstitial fluid for normal cellular functions. SLC20A2 variants have been found in patients with idiopathic basal ganglia calcification (IBGC), also known as Fahr’s disease or primary familial brain calcification (PFBC). Thus, disrupted Pi homeostasis is considered one of the major factors in the pathogenic mechanism of IBGC. In this paper, among the causative genes of IBGC, we focused specifically on PiT2, and its potential for a therapeutic target of IBGC.

Electrogenic Na+/[Formula: see text] cotransporter 1-A (NBCe1-A) in the proximal tubule regulates the acid/base balance and fluid volume homeostasis. From the National Center for Biotechnology Information dbSNP database, we identified the I551F variant of NBCe1-A, which showed reduced glycosylation, cell surface expression, and transport activity. We also found that the I551F variant can exert a dominant negative effect on wild-type NBCe1-A, suggesting its physiological significance.

BACKGROUND/AIMS: The osmolytes involved in the volume regulation of hyposmotically-swollen fish cells are well identified. However, if a coordination and adjustments of their fluxes are obvious, few studies have clearly illustrated these aspects. METHODS: Trout red blood cells volume variations were estimated from water contents obtained by a gravimetric method. Intracellular K+ and Na+ contents, and Cl- content of haemolysed cells were determined by photometry and colorimetry, respectively. The taurine contribution to cell volume regulation was calculated from the net changes of water, K+, Cl- and Na+ contents. The intracellular pH was calculated from the chloride distribution across the cells membranes according to the Donnan equilibrium. RESULTS: Cells responses to a rapid change (from 296 to 176 mOsm.kg-1) of the saline osmolality were examined in three conditions designed to not impact (Hypo. I) or to reduce the K+ (Hypo. II) and Cl- (Hypo. III) contributions to the volume regulation. Hypo. I condition caused an immediate increase in water content, followed by a 90 min. full regulation, concomitant with gradual lowering of K+ and Cl- contents and a surprising increase in Na+ content. Hypo. II and III conditions showed a partial and complete volume regulation, respectively. This was made possible by an increase in the taurine involvement. These experiments allowed to confirm that K+ and Cl- were released via KCl cotransport and by separate channels. The comparison of Hypo. I and III conditions led to the observation that the partially amiloride-sensitive Na+ influx is proportional to the taurine efflux; the latter being sustained mainly by a Na+/taurine cotransport. The Hypo. II condition was suitable for the (Na+/K+)ATPase activity inhibition. This effect could explain the observed lack of Na+ uptake, the consecutive depletion of intracellular taurine stock and the incomplete volume regulation. Finally, the results support the importance of taurine in pH control under Hypo. I (physiologic) condition. The alkalosis observed in Hypo. II and III conditions were the consequences of changes in the salines compositions, not of physiologic adjustments. CONCLUSION: The regulatory volume decrease process of trout RBCs is complex and adjustable through coordinated osmolytes movements. The obliged decrease in K+ and/or Cl- contributions stimulates taurine and Na+ pathways. This study highlights the importance of taurine as a compensatory variable in cell volume regulation and explains for the first time the significance of the Na+ uptake during this process

We report two pyridyl-linked benzimidazolyl hydrazones as HCl cotransporters that are 5 and 2 times superior to prodigiosin, a natural product whose transport efficiency has never been routed by synthetic molecules. These hydrazones provide a suitable HCl binding site through a cooperative protonation and chloride ion recognition. HCl transport by the most active compound induces lysosome deacidification. Viability assays confirmed that the compounds induce cytotoxicity toward human breast cancer MCF-7 cells but are relatively nontoxic toward noncancerous HEK293T cells.

Key points Extracellular space (ECS) rapid volume pulsation (RVP) accompanying epileptiform activity is described for the first time. Such RVP occurs robustly in several in vitro and in vivo mouse models of epileptiform activity. In the in vitro 4-aminopyridine model of epileptiform activity, RVP depends on the activity of the electrogenic Na+ /HCO3 - cotransporter (NBCe1). NBCe1 pharmacological inhibition suppresses RVP and epileptiform activity. Inhibition of changes in ECS volume may represent a useful target in epilepsy patients who are resistant to current treatments. Abstract The extracellular space (ECS) of the brain shrinks persistently by approximately 35% during epileptic seizures. Here we report the discovery of rapid volume pulsation (RVP), further transient drops in ECS volume which accompany events of epileptiform activity. These transient ECS contractions were observed in multiple mouse models of epileptiform activity both in vivo (Bicuculline Methiodide model) and in vitro (Hyaluronan synthase 3 knock-out, Picrotoxin, Bicuculline, and 4-Aminopyridine models). By using the probe transients quantification (PTQ) method we show that individual pulses of RVP shrank the ECS by almost 15% in vivo. In the 4-Aminopyridine in vitro model, the individual pulses of RVP shrank the ECS by more than 4%, and these transient changes were superimposed on a persistent ECS shrinkage of 36% measured with the real-time iontophoretic method. In this in vitro model, we investigated several channels and transporters that may be required for the generation of RVP and epileptiform activity. Pharmacological blockages of Na+ /K+ /2Cl- cotransporter type 1 (NKCC1), K+ /Cl- cotransporter (KCC2), the water channel Aquaporin-4 (AQP4) and inwardly-rectifying potassium channel 4.1 (Kir4.1) were ineffective in halting the RVP and the epileptiform activity. In contrast, pharmacological blockade of the electrogenic Na+ /HCO3 - cotransporter (NBCe1) by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminated both the RVP and the persistent ECS shrinkage. Importantly, this blocker also stopped the epileptiform activity. These results demonstrate that RVP is closely associated with epileptiform activity across several models of epileptiform activity and therefore the underlying mechanism could potentially represent a novel target for epilepsy management and treatment. This article is protected by copyright. All rights reserved.

Purpose of review Sodium-glucose cotransporter 2 (SGLT2) inhibitors are widely used antihyperglycemic drugs that show remarkable cardiorenal protective effects in patients with or without type 2 diabetes. Furthermore, they are effective among patients across a wide range of baseline renal and cardiac function. Numerous mechanisms have been evaluated to understand these remarkable clinical benefits. From an early stage, these agents were noted to affect the plasma lipid profile. Here we review lipid profile alterations attributable to SGLT2 inhibitors and also some mechanisms explored in model systems and human studies. Recent findings SGLT2 inhibitors given to patients with diabetes as monotherapy shift substrate utilization from carbohydrates to lipids, and have mild effects on the lipid profile. Increased LDL cholesterol appears to be associated with increased hepatic production and decreased catabolism. Increased HDL cholesterol and decreased triglycerides appear to be associated with improved insulin sensitivity and increased lipolysis. Lipid effects of SGLT2 inhibitors are further modulated by background therapy with other diabetes medications and statins. Summary The minor lipid profile alterations observed in patients treated with SGLT2 inhibitors are offset by the staggering range of beneficial pleiotropic mechanisms that likely explain the marked cardiorenal benefits of these agents.

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.

Mammalian cells take in d-glucose as an essential fuel as well as a carbon source. In contrast, l-glucose, the mirror image isomer of d-glucose, has been considered merely as a non-transportable/non-metabolizable control for d-glucose. We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a d-glucose analogue combining a fluorophore NBD at the C-2 position, is useful as a tracer for monitoring d-glucose uptake through glucose transporters (GLUTs) into mammalian cells. To more precisely evaluate the stereoselectivity of 2-NBDG uptake, we developed an l-glucose analogue 2-NBDLG, the mirror-image isomer of 2-NBDG. Interestingly, 2-NBDLG was taken up into mouse insulinoma MIN6 cells showing nuclear heterogeneity, a cytological feature of malignancy, while remaining MIN6 cells only exhibited a trace amount of 2-NBDLG uptake. The 2-NBDLG uptake into MIN6 cells was abolished by phloretin, but persisted under blockade of major mammalian glucose transporters. Unfortunately, however, no such uptake could be detected in other tumor cell lines. Here we demonstrate that human osteosarcoma U2OS cells take in 2-NBDLG in a phloretin-inhibitable manner. The uptake of 2-NBDG, and not that of 2-NBDLG, into U2OS cells was significantly inhibited by cytochalasin B, a potent GLUT inhibitor. Phloretin, but neither phlorizin, an inhibitor of sodium-glucose cotransporter (SGLT), nor a large amount of d/l-glucose, blocked the 2-NBDLG uptake. These results suggest that a phloretin-inhibitable, non-GLUT/non-SGLT, possibly non-transporter-mediated yet unidentified mechanism participates in the uptake of the fluorescent l-glucose analogue in two very different tumor cells, the mouse insulinoma and the human osteosarcoma cells.

Hydrogen sulfide (H2S) has emerged as a novel gaseous signal molecule with multifarious effects on seed germination, plant growth, development, and physiological processes. Due to its dominant role in plant stress tolerance and cross-adaptation, it is getting more attention nowadays, although it has been largely referred as toxic and environmental hazardous gas. In this review work, we are highlighting the importance of H2S as an essential gaseous molecule to help in signaling, metabolism, and stress tolerance in plants. Firstly, production of H2S from different natural and artificial sources were discussed with its transformation from sulfur (S) to sulfate (SO42−) and then to sulfite (SO32−). The importance of different kinds of transporters that helps to take SO42− from the soil solution was presented. Mainly, these transporters are SULTRs (H+/SO42− cotransporters) and multigene family encodes them. Furthermore, these SULTRs have LAST (Low affinity transport proteins), HAST (High affinity transport proteins), vacuole transporters, and plastid transporters. Since it is well known that there is strong relationship between SO42− and synthesis of hydrogen sulfide or dihydrogen sulfide or sulfane in plant cells. Thus, cysteine (Cys) metabolism through which H2S could be generated in plant cell with the role of different enzymes has been presented. Furthermore, H2S in interaction with other molecules could help to mitigate biotic and abiotic stress. Based on this review work, it can be concluded that H2S has potential to induce cross-adaptation to biotic and abiotic stress; thus, it is recommended that it should be considered in future studies to answer the questions like what are the receptors of H2S in plant cell, where in plants the physiological concentration of H2S is high in response to multiple stress and how it induces cross-adaptation by interaction with other signal molecules.

Intestinal absorption of phosphate is stimulated by 1,25(OH)$_{2}$ vitamin D$_{3.}$ At least two distinct mechanisms underlie phosphate absorption in the gut, an active transcellular transport requiring the Na$^{+}$ /phosphate cotransporter NaPi-IIb/Slc34a2, and a poorly characterized paracellular passive pathway. 1,25(OH)$_{2}$ vitamin D$_{3}$ stimulates NaPi-IIb expression and function and loss of NaPi-IIb reduces intestinal phosphate absorption. However, it is remains unknown whether NaPi-IIb is the only target for hormonal regulation by 1,25(OH)$_{2}$ vitamin D$_{3}$ . Here we compared the effects of intraperitoneal administration of 1,25(OH)$_{2}$ vitamin D$_{3}$ (2 days, once per day) in wild type and intestinal-specific Slc34a2 deficient mice, and analyzed trans- vs paracellular routes of phosphate absorption. We found that treatment stimulated active transport of phosphate only in jejunum of wild type mice, though NaPi-IIb protein expression was upregulated in jejunum and ileum . In contrast, 1,25(OH)$_{2}$ vitamin D$_{3}$ administration had no effect in Slc34a2 deficient mice, suggesting that the hormone specifically regulates NaPi-IIb expression. In both groups, 1,25(OH)$_{2}$ vitamin D$_{3}$ elicited the expected increase of plasma FGF23 and reduction of PTH. Treatment resulted in hyperphosphaturia (and hypercalciuria) in both genotypes, though mice remained normophosphatemic. While increased intestinal absorption and higher FGF23 can trigger the hyperphosphaturic response in wild types, only the second one can explain the renal response in Slc34a2 deficient mice. Thus, 1,25(OH)$_{2}$ vitamin D$_{3}$ stimulates intestinal phosphate absorption by acting on the active transcellular pathway mostly mediated by NaPi-IIb while the paracellular pathway appears not to be affected. Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi-IIb/Slc34a2 and a poorly characterized passive/paracellular pathway. Intestinal phosphate absorption and expression of NaPi-IIb are stimulated by 1,25(OH)$_{2}$ vitamin D$_{3}$ but whether NaPi-IIb is the only target under hormonal control remains unknown. We report that administration of 1,25(OH)$_{2}$ vitamin D$_{3}$ to wild type mice results in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes. Consequently, treatment failed to alter phosphate transport in intestinal-depleted Slc34a2 mice. In both genotypes, 1,25(OH)$_{2}$ vitamin D$_{3}$ induced similar hyperphosphaturic responses and changes in FGF23 and PTH. While urinary phosphate loss induced by administration of 1,25(OH)$_{2}$ vitamin D$_{3}$ did not alter plasma phosphate, further studies should investigate whether chronic administration would lead to phosphate imbalance in mice with reduced active intestinal absorption. This article is protected by copyright. All rights reserved.

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 sodium-phosphate cotransporter NPT2a plays a key role in the reabsorption of filtered phosphate in proximal renal tubules, thereby critically contributing to phosphate homeostasis. Inadequate urinary phosphate excretion can lead to severe hyperphosphatemia as in tumoral calcinosis and chronic kidney disease (CKD). Pharmacological inhibition of NPT2a may therefore represent an attractive approach for treating hyperphosphatemic conditions. The NPT2a-selective small-molecule inhibitor PF-06869206 was previously shown to reduce phosphate uptake in human proximal tubular cells in vitro. Here, we investigated the acute and chronic effects of the inhibitor in rodents and report that administration of PF-06869206 was well tolerated and elicited a dose-dependent increase in fractional phosphate excretion. This phosphaturic effect lowered plasma phosphate levels in WT mice and in rats with CKD due to subtotal nephrectomy. PF-06869206 had no effect on Npt2a-null mice, but promoted phosphate excretion and reduced phosphate levels in normophophatemic mice lacking Npt2c and in hyperphosphatemic mice lacking Fgf23 or Galnt3. In CKD rats, once-daily administration of PF-06869206 for 8 weeks induced an unabated acute phosphaturic and hypophosphatemic effect, but had no statistically significant effect on FGF23 or PTH levels. Selective pharmacological inhibition of NPT2a thus holds promise as a therapeutic option for genetic and acquired hyperphosphatemic disorders.

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

Objective To review the incidence, risk factors, prevention, and management of genital mycotic infections (GMIs) associated with sodium-glucose cotransporter 2 (SGLT2) inhibitors. Data Sources A literature search of PubMed and Reactions Weekly was performed in February 2020 with updated searches monthly through July 2020 to identify relevant data regarding SGLT2 inhibitors and GMIs. Manufacturers of each agent were contacted, and clinical practice guidelines were consulted. Study Selection and Data Extraction All available literature was evaluated for inclusion based on relevance to the research question, timeliness of the publication, validity, and impact on current practice. A date limit was not set; however, publications from 2010 to July 2020 were prioritized. Data Synthesis The 3- to 4-fold increased incidence of GMIs is considered a classwide effect of SGLT2 inhibitors. Female sex and a prior history of GMIs are factors associated with the highest risk, whereas circumcised males are at the lowest risk of SGLT2 inhibitor–induced GMI. Personal hygiene advice can reduce the infection risk. When candidiasis occurs, it is often mild and responsive to treatment and often does not require discontinuation of the medication. Relevance to Patient Care and Clinical Practice This narrative review can assist in shared decision-making discussions with patients who may benefit from SGLT2 inhibitors and provides guidance for health care professionals managing SGLT2 inhibitor–associated GMIs. Conclusions SGLT2 inhibitors predispose patients to developing mild GMIs. Strong consideration should be given to avoid SGLT2 inhibitors in female patients with a history of severe, recurrent infections. Preventive strategies are optimized diabetes management and personal hygiene advice.

We herein report the clinical course of a 56-year-old Japanese patient with slowly progressive type 1 diabetes mellitus, metabolic syndrome, non-alcoholic fatty liver disease, and severe insulin resistance. The patient’s intravenous glucose tolerance test indicated marked reductions in insulin sensitivity and endogenous insulin secretion. Accordingly, administration of ipragliflozin l-proline, a sodium-glucose cotransporter 2 inhibitor, promoted improvements in insulin sensitivity and blood glucose levels, as well as a decrease in visceral fat, improvement in dyslipidemia, and decrease in hepatic lipid content, suggesting the potential efficacy of sodium-glucose cotransporter 2 inhibitors for obese patients with type 1 diabetes mellitus exhibiting insulin resistance.

The porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1−/− and wild type (WT) mice. Hydrostatic pressure (HP) in lens surface cells was determined using a manometer-coupled microelectrode approach. The TRPV1 agonist capsaicin (100 nM) caused a transient HP increase in WT lenses that peaked after ∼30 min and then returned toward baseline. Capsaicin did not cause a detectable change of HP in TRPV1−/− lenses. The NKCC inhibitor bumetanide prevented the HP response to capsaicin in WT lenses. Potassium transport was examined by measuring Rb+ uptake. Capsaicin increased Rb+ uptake in cultured WT lens epithelial cells but not in TRPV1−/− cells. Bumetanide, A889425, and the Akt inhibitor Akti prevented the Rb+ uptake response to capsaicin. The bumetanide-sensitive (NKCC-dependent) component of Rb+ uptake more than doubled in response to capsaicin. Capsaicin also elicited rapid (−/− cells. HP recovery was shown to be absent in TRPV1−/− lenses exposed to hyperosmotic solution. Bumetanide and Akti prevented HP recovery in WT lenses exposed to hyperosmotic solution. Taken together, responses to capsaicin and hyperosmotic solution point to a functional role for TRPV1 channels in mouse lens. Lack of NKCC1 phosphorylation and Rb+ uptake responses in TRPV1−/− mouse epithelium reinforces the notion that a hyperosmotic challenge causes TRPV1-dependent NKCC1 activation. The results are consistent with a role for the TRPV1-activated signaling pathway leading to NKCC1 stimulation in lens osmotic homeostasis.

The influence of dietary vitamin D3 (VD3) levels on growth, bone performance, and duodenal type IIb sodium-dependent phosphate cotransporter (NaPi-IIb) genes in broiler chicken were studied. One-day-old male Ross308 broilers (n = 432) were allocated into 6 treatment groups with each group consisting of 6 cage pens. Each treatment group received diet containing different amounts of VD3 (80, 200, 500, 1,250, 3,125, or 7,813 IU per kg of diet) from a day-old to 31 D of age. Dietary available phosphorus and calcium were kept the same across all treatments in each phase. At 14 D, influence of VD3 on BW gain was found in the birds that received VD3 of 3,125 IU/kg and 200 IU/kg (P

Sodium-glucose cotransporter 2(SGLT2) inhibitors show prominent renal protective effect in diabetic kidney disease (DKD), anti-inflammatory effect being one of its key mechanisms. Over-activation of the complement system, a crucial part of innate immunity, plays an important role in DKD. We aimed to investigate the effect of SGLT2 inhibitors on alleviating complement over-activation in DKD. Db/db mice were randomly divided into two groups, with 7 mice in each group treated with dapagliflozin and vehicle respectively, and 7 mice in m/m mice group. Laboratory and renal pathological parameters were evaluated. Mouse proximal tubular epithelial cells (MPTECs) were cultured and treated with high glucose. Dapagliflozin and dimethyloxallyl glycine (DMOG) were added as conditional treatment. Dapagliflozin-treated db/db mice showed significantly lower urinary albumin than vehicle-treated ones. Besides typical glomerular and tubulointerstitial injury, both C3b and membrane attack complex (MAC) depositions were significantly attenuated in dapagliflozin-treated db/db mice. The expression of complement receptor type 1-related protein y (Crry), a key complement regulator which inhibits complement over-activation, was significantly upregulated by dapagliflozin. Dapagliflozin-mediated Crry upregulation was associated with inhibition of HIF-1α accumulation under high glucose. When HIF-1α expression was stabilized by DMOG, the protective effect of dapagliflozin via upregulating Crry was blocked. In conclusion, dapagliflozin could attenuate complement over-activation in diabetic mice via upregulating Crry, which is associated with the suppression of HIF-1α accumulation in MPTECs.

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.

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport., The mechanisms involved in SLC4-mediated ion transport are still under debate. Here, the authors present a cryoEM structure of the Sodium-driven Chloride/Bicarbonate Exchanger NDCBE, which - together with computational modeling and mutagenesis - reveals molecular determinants of ion transport by SLC4.

Sodium-glucose cotransporter (SGLT) 2 inhibitors reduce glucose reabsorption in the kidney, increase glucosuria, and improve glycemia. Besides glycemic efficacy, the class also lowers risk of cardiovascular and renal disease.The authors describe late phase trials of empagliflozin, canagliflozin, dapagliflozin, and ertugliflozin. Safety and efficacy endpoints in monotherapy, combination therapy, cardiovascular, and renal outcomes trials have been identified and presented.SGLT2 inhibitors appear to be safe and effective agents that improve glycemia when used alone or in combination with any other approved antihyperglycemic medications. Other beneficial effects include reductions in body weight and blood pressure, improvements in renal outcomes, all-cause mortality, cardiovascular mortality, and worsening heart failure.

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.

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.

Cu(I) P-type ATPases are transmembrane primary active ion pumps that catalyze the extrusion of copper ions across cellular membranes. Their activity is critical in controlling copper levels in all kingdoms of life. Biochemical and structural characterization established the structural framework by which Cu-pumps perform their function. However, the details of the overall mechanism of transport (uniporter vs. cotransporter) and electrogenicity still remain elusive. In this work, we developed a platform to reconstitute the model Cu(I)-pump from E. coli (EcCopA) in artificial lipid bilayer small unilamellar vesicles (SUVs) to quantitatively characterize the metal substrate, putative counterions and charge translocation. By encapsulating in the liposome lumen fluorescence detector probes (CTAP-3, pyranine and oxonol VI) responsive to diverse stimuli (Cu(I), pH and membrane potential), we correlated substrate, secondary-ion translocation and charge movement events in EcCopA proteoliposomes. This platform centered on multiple fluorescence reporters allowed study of the mechanism and translocation kinetic parameters in real-time for wild-type EcCopA and inactive mutants. The maximal initial Cu(I) transport rate of 165 nmol Cu(I) mg(–1) min(–1) and K(M,Cu(I)) = 0.15 ± 0.07 μM was determined with this analysis. We reveal that Cu(I) pumps are primary-active uniporters and electrogenic. The Cu(I) translocation cycle does not require proton counter-transport resulting in electrogenic generation of transmembrane potential upon translocation of one Cu(I) per ATP hydrolysis cycle. Thus, mechanistic differences between Cu(I) pumps and other better characterized P-type ATPases are discussed. The platform opens the venue to study translocation events and mechanisms of transport in other transition metal P-type ATPase pumps.

Daily dietary potassium (K+) intake may be as large as the extracellular K+pool. To avoid acute hyperkalemia, rapid removal of K+from the extracellular space is essential. This is achieved by translocating K+into cells and increasing urinary K+excretion. Emerging data now indicate that the renal thiazide-sensitive NaCl cotransporter (NCC) is critically involved in this homeostatic kaliuretic response. This suggests that the early distal convoluted tubule (DCT) is a K+sensor that can modify sodium (Na+) delivery to downstream segments to promote or limit K+secretion. K+sensing is mediated by the basolateral K+channels Kir4.1/5.1, a capacity that the DCT likely shares with other nephron segments. Thus, next to K+-induced aldosterone secretion, K+sensing by renal epithelial cells represents a second feedback mechanism to control K+balance. NCC’s role in K+homeostasis has both physiological and pathophysiological implications. During hypovolemia, NCC activation by the renin-angiotensin system stimulates Na+reabsorption while preventing K+secretion. Conversely, NCC inactivation by high dietary K+intake maximizes kaliuresis and limits Na+retention, despite high aldosterone levels. NCC activation by a low-K+diet contributes to salt-sensitive hypertension. K+-induced natriuresis through NCC offers a novel explanation for the antihypertensive effects of a high-K+diet. A possible role for K+in chronic kidney disease is also emerging, as epidemiological data reveal associations between higher urinary K+excretion and improved renal outcomes. This comprehensive review will embed these novel insights on NCC regulation into existing concepts of K+homeostasis in health and disease.

Phosphate plays a critical role in many vital cellular processes. Deviations from normal serum phosphate levels, including alterations in the extracellular phosphate/pyrophosphate ratio, can cause severe consequences, such as ectopic calcification. Cellular phosphate levels are tightly controlled by sodium phosphate cotransporters, underscoring their importance in cellular physiology. The role of sodium phosphate cotransporters in ectopic calcification requires further elucidation, taking into account their important role in the control of intracellular phosphate levels and the synthesis of ATP, the main source of extracellular pyrophosphate (a potent endogenous inhibitor of calcification). In this review, we discuss the roles of phosphate and pyrophosphate homeostasis in ectopic calcification, with a specific focus on phosphate transporters. We concentrate on the five known sodium-dependent phosphate transporters and review their localisation and regulation by external factors, and the effects observed in knockout studies and in naturally occurring mutations.

Low Na+intake activates aldosterone signaling, which increases renal Na+reabsorption through increased apical activity of the NaCl cotransporter (NCC) and the epithelial Na+channel (ENaC). Na+transporter proteins are excreted in urine as an integral part of cell-derived extracellular vesicles (uEVs). It was hypothesized that Na+transport protein levels in uEVs from healthy humans reflect their physiological regulation by aldosterone. Urine and plasma samples from 10 healthy men (median age: 22.8 yr) were collected after 5 days on a low-Na+(70 mmol/day) diet and 5 days on a high-Na+(250 mmol/day) diet. uEVs were isolated by ultracentrifugation and analyzed by Western blot analysis for EV markers (CD9, CD63, and ALIX), transport proteins (Na+-K+-ATPase α1-subunit, NCC, ENaC α- and γ-subunits, and aquaporin 2), and the ENaC-cleaving protease prostasin. Plasma renin and aldosterone concentrations increased during the low-Na+diet. uEV size and concentration were not different between diets by tunable resistive pulse sensing. EV markers ALIX and CD9 increased with the low-Na+diet, whereas CD63 and aquaporin 2 excretion were unchanged. Full-length ENaC γ-subunits were generally not detectable in uEVs, whereas ENaC α-subunits, NCC, and phosphorylated NCC were consistently detected but not changed by Na+intake. Prostasin increased with low Na+in uEVs. uEV excretion of transporters was not correlated with blood pressure, urinary Na+and K+excretion, plasma renin, or aldosterone. In conclusion, apical Na+transporter proteins and proteases were excreted in uEVs, and while the excretion rate and size of uEVs were not affected, EV markers and prostasin increased in response to the low-Na+diet.

Sodium bicarbonate has long been used to treat chronic kidney disease. It has been demonstrated to slow the decline in glomerular filtration rate in chronic kidney disease patient; however, the mechanisms are not completely understood. We hypothesized that NaHCO 3 dilates afferent arterioles (Af-Art) by stimulating nitric oxide (NO) release mediated by the Na + /HCO 3 − cotransporter (NBC) contributing to the elevation in glomerular filtration rate. Isolated microperfused mouse renal Af-Art, preconstricted with norepinephrine (1 µmol/L), dilated 45±2% (n=6, P 3 (44 mmol/L). Whereas, NaCl solution containing the same Na + concentration was not effective. The mRNA for NBCn1 and NBCe1 were detected in microdissected Af-Art using reverse transcription–polymerase chain reaction and quantitative polymerase chain reaction. The Af-Art intracellular pH measured with 2′,7′-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein, acetoxymethyl ester increased significantly by 0.29±0.02 (n=6; P 3 , which was blunted by N-cyanosulphonamide compound (S0859) that is an inhibitor of the NBC family. After clamping the intracellular pH with 10 μM nigericin, changing the bath solution pH from 7.4 to 7.8 still dilates the Af-Art by 53±4% (n=7; P P 3 increased NO generation in Af-Art by 19±4% (n=5; P P 3 -induced increases in NO generation and vasodilation. We conclude that NBCn1 and NBCe1 are expressed in Af-Art and that NaHCO 3 dilates Af-Art via NBCs mediated by NO that increases the glomerular filtration rate.

Effects of dietary non-phytate phosphorus (nPP) restriction on growth and duodenal type IIb sodium-dependent phosphate cotransporter (NaPi-IIb) genes were observed. A total of 432 one-day old Cobb500 male broiler chickens in 36 cage pens were divided into 6 groups with each group containing 6 pens. Each group was treated with one of the diets containing 0.33, 0.37, 0.41, 0.45, 0.49, and 0.53% of nPP up to 14 D. During 15 to 31 D, birds were treated with one of the diets containing 0.23, 0.27, 0.31, 0.35, 0.39, and 0.43% of nPP. Level of Ca was kept the same across all treatments. Dietary nPP level influenced (P < 0.001) weight gain and feed intake in both growth phases, whereas effect on feed per gain ratio was seen only in the second phase. Toe ash, tibia ash, and tibia breaking strength responded to treatments (P < 0.01) at 14 D. Only tibia ash content was significantly improved (P < 0.001) at 31 D. Growth and bone parameters linearly improved with an increase in dietary nPP content (P < 0.05). Above dietary nPP 0.41% and 0.31% for first phase and second phase, respectively, no significant improvement was seen. Duodenal NaPi-IIb mRNA overexpressed with a decrease in dietary nPP in both phases (P < 0.05). Relative expression of NaPi-IIb in lowest nPP group were 2.2 folds higher in the first phase and 3.6 folds higher in the second phase compared to respective highest nPP groups of each phase. No significant change in NaPi-IIb expression was seen above 0.37% of dietary nPP for 14 D and 0.31% of dietary nPP for 31 D. Dietary requirements of nPP 0.41% for 0 to 14 D and 0.31% for 15 to 31 D were adequate for optimal growth and bone parameters. This study fills the gap in understanding of intestinal NaPi-IIb expression in response to dietary nPP restriction in broilers older than 21 D of age.

This study investigated the cotransport behaviors of graphene oxides (GO) and reduced graphene oxides (RGO) with bisphenol A (BPA) in porous media in both NaCl (1 and 10 mmol/L) and CaCl2 solutions (0.5 and 1.5 mmol/L) at pH 6.5. Both bare and iron oxides-coated quartz sand were employed as porous media in present study. We found that under all examined solution conditions, the presence of BPA (100 µ/L) did not have obvious influence on the transport of both GO and RGO (8 mg/L as TOC) in both bare and iron oxides-coated quartz sand. Although the dissolved BPA was the major form dominating the transport behaviors of total BPA in the presence of GO/RGO, yet the GO/RGO-associated BPA (due to the adsorption of BPA onto GO/RGO surfaces) also had some contribution to the transport of total BPA in the presence of GO/RGO in two types of porous media. Overall, due to the different transport behaviors of GO and RGO under different solution conditions, we found that the presence of GO/RGO decreased the transport of total BPA under all examined solution conditions in two types of porous media with the smallest decrease in 1 mmol/L NaCl solutions and the largest in 1.5 mmol/L CaCl2 solutions. The results of this study clearly indicated that when BPA was co-present with GO/RGO, the transport behaviors of GO/RGO in porous media would have great influences on the fate and transport of BPA in natural environments due to their adsorption onto GO/RGO.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl− cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

Cellular chloride transport has a fundamental role in cell volume regulation and renal salt handling. Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Thus, we identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume. Highlights: Protein kinase SYK phosphorylates NKCC2 and KCC3 at an N-terminal tyrosine residue; Cellular SYK activity modulates the expression of NKCC2 and KCC3 at the cell surface; SYK activity modulates cell surface expression of NKCC2 and KCC3 in opposite ways; SYK-modulated cell surface expression of NKCC2 contributes to cell-volume regulation. This work was supported by Fundação para a Ciência e Tecnologia (FCT) [grants PTDC/SAU-ORG/119782/2010 and PTDC/BIA-CEL/28408/2017 to PJ, grant UID/MULTI/04046/2019 to the research unit BioISI, and fellowship SFRH/BD/52488/2014 from the BioSYS PhD programme PD65-2012 to CAL]. The authors acknowledge the following colleagues for providing reagents used in this study: K. Mutig, Charité, Berlin, Germany; Maria J. Valente, REQUIMTE, Porto, Portugal; Dimitar G. Efremov, ICGEB, Rome, Italy. info:eu-repo/semantics/publishedVersion

How taste buds detect NaCl remains poorly understood. Among other problems, applying taste-relevant concentrations of NaCl (50–500 mm) onto isolated taste buds or cells exposes them to unphysiological (hypo/hypertonic) conditions. To overcome these limitations, we used the anterior tongue of male and female mice to implement a slice preparation in which fungiform taste buds are in a relatively intact tissue environment and stimuli are limited to the taste pore. Taste-evoked responses were monitored using confocal Ca(2+) imaging via GCaMP3 expressed in Type 2 and Type 3 taste bud cells. NaCl evoked intracellular mobilization of Ca(2+) in the apical tips of a subset of taste cells. The concentration dependence and rapid adaptation of NaCl-evoked cellular responses closely resembled behavioral and afferent nerve responses to NaCl. Importantly, taste cell responses were not inhibited by the diuretic, amiloride. Post hoc immunostaining revealed that >80% of NaCl-responsive taste bud cells were of Type 2. Many NaCl-responsive cells were also sensitive to stimuli that activate Type 2 cells but never to stimuli for Type 3 cells. Ion substitutions revealed that amiloride-insensitive NaCl responses depended on Cl(−) rather than Na(+). Moreover, choline chloride, an established salt taste enhancer, was equally effective a stimulus as sodium chloride. Although the apical transducer for Cl(−) remains unknown, blocking known chloride channels and cotransporters had little effect on NaCl responses. Together, our data suggest that chloride, an essential nutrient, is a key determinant of taste transduction for amiloride-insensitive salt taste. SIGNIFICANCE STATEMENT Sodium and chloride are essential nutrients and must be regularly consumed to replace excreted NaCl. Thus, understanding salt taste, which informs salt appetite, is important from a fundamental sensory perspective and forms the basis for interventions to replace/reduce excess Na(+) consumption. This study examines responses to NaCl in a semi-intact preparation of mouse taste buds. We identify taste cells that respond to NaCl in the presence of amiloride, which is significant because much of human salt taste also is amiloride-insensitive. Further, we demonstrate that Cl(−), not Na(+), generates these amiloride-insensitive salt taste responses. Intriguingly, choline chloride, a commercial salt taste enhancer, is also a highly effective stimulus for these cells.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed significant effects in patients with diabetes or metabolic syndrome (MetS) with high cardiovascular risk. Although the increased intracellular Zn2+ level ([Zn2+]i), oxidative stress, and altered cardiac matrix metalloproteinases (MMPs) in diabetic cardiomyopathy can intersect with different signaling pathways, the exact mechanisms are not known yet. Since either MMPs or SGLT2 have important roles in cardiac-fibrosis under hyperglycemia, we aimed to examine the role of SGLT2 inhibitor dapagliflozin (DAP) on cardiac Zn2+-transporters responsible for [Zn2+]i-regulation, comparison to insulin (INS), together with MMP levels and systemic oxidative stress status in MetS-rats. High-carbohydrated diet-induced MetS-rats received DAP or INS for 2 weeks. DAP but not INS in MetS-rats significantly decreased high blood-glucose levels, while both treatments exerted benefits on increased total oxidative status and decreased total antioxidant status in MetS-rat plasma as well as in heart tissue. Protein levels of Zn2+-transporters, responsible for Zn2+-influx into cytosol, ZIP7 and ZIP14 were increased with significant decrease in ZIP8 of MetS-rat cardiomyoctes, while Zn2+-transporters, responsible for cytosolic Zn2+-efflux, ZnT7 was decreased with no change in ZnT8. Both treatments induced significant beneficial effects on altered ZIP14, ZIP8, and ZnT7 levels. Furthermore, both treatments exerted benefits on depressed gelatin-zymography and protein expression levels of MMP-2 and MMP-9 in MetS-rat ventricular cardiomyocytes. The direct effect of DAP on heart was also confirmed with measurements of left ventricular developed pressure. Overall, we showed that DAP has important antioxidant-like cardio-protective effects in MetS-rats, similar to INS-effect, affecting Zn2+-regulation via Zn2+-transporters, MMPs, and oxidative stress. Therefore one can suggest that SGLT2 inhibitors can be new therapeutic agents for cardio-protection not only in hyperglycemia but also in failing heart.

Hydrogen sulphide (H2 S) is emerging as an important signalling molecule involved in plant resistance to various stresses. However, the underlying mechanism of H2 S in aluminium (Al) resistance and the crosstalk between H2 S and nitric oxide (NO) in Al stress signalling remain elusive. Citrate secretion is a wide-spread strategy for plants against Al toxicity. Here, two citrate transporter genes, GmMATE13 and GmMATE47, were identified and characterized in soybean. Functional analysis in Xenopus oocytes and transgenic Arabidopsis showed that GmMATE13 and GmMATE47 mediated citrate exudation and enhanced Al resistance. Al treatment triggered H2 S generation and citrate exudation in soybean roots. Pretreatment with an H2 S donor significantly elevated Al-induced citrate exudation, reduced Al accumulation in root tips, and alleviated Al-induced inhibition of root elongation, whereas application of an H2 S scavenger elicited the opposite effect. Furthermore, H2 S and NO mediated Al-induced GmMATE expression and plasma membrane (PM) H+ -ATPase activity and expression. Further investigation showed that NO induced H2 S production by regulating the key enzymes involved in biosynthesis and degradation of H2 S. These findings indicate that H2 S acts downstream of NO in mediating Al-induced citrate secretion through the upregulation of PM H+ -ATPase-coupled citrate transporter cotransport systems, thereby conferring plant resistance to Al toxicity.

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.

KCC2 is a brain specific chloride–potassium cotransporter affecting neuronal development including migration and cellular maturation. It modulates chloride homeostasis influencing the switch of GABA from depolarizing to hyperpolarizing, which contributes to the cues that influence the termination of neuronal migration. The expression of KCC2 during migration of interneurons, therefore, correlates with the ability of these cells to respond to GABA as a stop signal. Manipulation of KCC2 in development can affect various aspects of migrating neurons, including the speed. We describe the effect of KCC2 downregulation and inhibition on features of migrating interneurons of normal ferret kits and those treated with methylazoxymethanol acetate, which increases KCC2. Treatment of organotypic cultures with Bisphenol A, an environmental toxin that alters gene expression, also downregulates KCC2 protein. In organotypic slices treated with the KCC2 antagonist VU0240551, chloride imaging shows inhibition of KCC2 via blockade of chloride flux. Time-lapse video imaging of organotypic cultures treated with either drug, shows a significant increase in the average speed, step size, and number of turns made by migrating neurons leaving the ganglionic eminence. Our findings demonstrate the harmful effect of environmental toxins on brain development and potential consequences in the pathogenesis of neurodevelopmental disorders.

Ipragliflozin, a selective sodium glucose cotransporter 2 (SGLT2) inhibitor, is used for the treatment of type 2 diabetes mellitus. To date, the only known in vitro pharmacological characteristic of ipragliflozin is its selectivity for SGLT2 over SGLT1, which was previously reported by our group. Therefore, in this study, we investigated other in vitro pharmacological characteristics of ipragliflozin and compared them with those of phlorizin, a naturally occurring SGLT inhibitor. Selectivity of ipragliflozin and phlorizin for human (h) SGLT2 over hSGLT3, hSGLT4, hSGLT5, hSGLT6 and hSodium/myo-inositol (MI) cotransporter 1 (hSMIT1) was examined in Chinese hamster ovary (CHO) cells overexpressing each transporter using specific radio-ligands. Ipragliflozin had higher selectivity for hSGLT2 than other hSGLTs. Phlorizin showed lower selectivity for hSGLT2 compared to ipragliflozin. Studies using CHO cells overexpressing hSGLT2 demonstrated that both ipragliflozin and phlorizin competitively inhibited SGLT2-mediated methyl-α-D-glucopyranoside (AMG) uptake with an inhibitory constant (Ki) of 2.28 and 20.2 nM, respectively. Ipragliflozin, but not phlorizin, inhibited hSGLT2 in a wash-resistant manner, suggesting that binding of ipragliflozin to hSGLT2 was persistent. These data demonstrate that ipragliflozin is a competitive inhibitor of SGLT2, has high selectivity for SGLT2 over not only SGLT1 but also other SGLT family members, and binds persistently to hSGLT2.

Fructose consumption has increased because of widespread use of high-fructose corn syrup by the food industry. Renal proximal tubules are thought to reabsorb fructose. However, fructose reabsorption (Jfructose) by proximal tubules has not yet been directly demonstrated, nor the effects of dietary fructose on Jfructose. This segment expresses Na+- and glucose-linked transporters (SGLTs) 1, 2, 4, and 5 and glucose transporters (GLUTs) 2 and 5. SGLT4 and -5 transport fructose, but SGLT1 and -2 do not. Knocking out SGLT5 increases urinary fructose excretion. We hypothesize that Jfructosein the S2 portion of the proximal tubule is mediated by luminal entry via SGLT4/5 and basolateral exit by GLUT2 and that it is enhanced by a fructose-enriched diet. We measured Jfructoseby proximal straight tubules from rats consuming either tap water (Controls) or 20% fructose (FRU). Basal Jfructosein Controls was 14.1 ± 1.5 pmol·mm−1·min−1. SGLT inhibition with phlorizin reduced Jfructoseto 4.9 ± 1.4 pmol·mm−1·min−1( P < 0.008), whereas removal of Na+diminished Jfructoseby 86 ± 5% ( P < 0.0001). A fructose-enriched diet increased Jfructosefrom 12.8 ± 2.5 to 19.3 ± 0.5 pmol·mm−1·min−1, a 51% increase ( P < 0.03). Using immunofluorescence, we detected luminal SGLT4 and SGLT5 and basolateral GLUT2; GLUT5 was undetectable. The expression of apical transporters SGLT4 and SGLT5 was higher in FRU than in Controls [137 ± 10% ( P < 0.01) and 38 ± 14% ( P < 0.04), respectively]. GLUT2 was also elevated by 88 ± 27% ( P < 0.02) in FRU. We conclude that Jfructoseby proximal tubules occurs primarily via Na+-linked cotransport processes, and a fructose-enriched diet enhances reabsorption. Transport across luminal and basolateral membranes is likely mediated by SGLT4/5 and GLUT2, respectively.

Biochar widely applied to soil can influence microbial community composition and participate in extracellular electron transfer (EET). However, little is known about the cotransport behaviors of bacteria and biochar in aquifer and soil-water environments, which can affect the fate and application performance of biochar. In this study, we found that in comparison to their individual transport behaviors, the mobilities of cotransporting Shewanella oneidensis MR-1 and biochar colloid (BC) were significantly inhibited. The decreasing colloidal mobilities at higher ionic strengths signified the importance of electrostatic interaction between cell and BC in cotransport. Moreover, the less suppressed cotransport of BC and mutants defective of EET and the elevated inhibition effects on cotransport by adding exogenous electron donor suggested the importance of EET. Difference in cotransport behavior was also observed with BC having different redox states. Compared with oxidized BC, reduced BC with higher hydrophobicity led to easier aggregation with cell and higher retention in column. More importantly, MR-1 exhibited EET-dependent taxis towards biochar, which also contributed to the enhanced heteroaggregation and decreased mobilities of cell and biochar. Our results highlight that metabolic activities of microbes towards abiotic colloids cannot be neglected when assessing their transport behaviors, especially in subsurface environments abounded with redox-active inorganic particles and microbes performing extracellular respiration.

Hypertension is an important target for drug discovery. We have focused on the with-no-lysine kinase (WNK)-oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)-NaCl cotransporter (NCC) signal cascade as a potential target, and we previously developed a screening system for inhibitors of WNK-OSR1/SPAK-NCC signaling. Herein we used this system to examine the structure-activity relationship (SAR) of salicylanilide derivatives as SPAK kinase inhibitors. Structural design and development based on our previous hit compound, aryloxybenzanilide derivative 2, and the veterinary anthelmintic closantel (3) led to the discovery of compound 10 a as a potent SPAK inhibitor with reduced toxicity. Compound 10 a decreased the phosphorylation level of NCC in mouse kidney in vivo, and appears to be a promising lead compound for a new class of antihypertensive drugs.

Background and Aims The emergence of positive data on the use of sodium glucose co-transporter inhibitors (SGLT2i) in the last several years, begged the question of whether their positive outcomes can be seen in kidney transplant recipients, as they have the same and even more pronounced cardiovascular risk factors than the general population and in addition, we got better in improving graft and patients survival in the short term , but we lack the tools to improve long term patients and graft survival where so many patients die from cardiovascular disease with a functioning graft or lose their graft from chronic changes and chronic antibody mediated rejection with difficult to control blood pressure and proteinuria For these reasons we need more powerful tools , like the SGLT2i bearing in mind the unique side effects that might be amplified in kidney transplant recipients receiving immunosuppression like urinary tract infection, and the dip in serum creatinine. Method We collected data retrospectively from transplant records of patients with type II diabetes mellitus (T2D) or post-transplant diabetes mellitus (PTDM) (n=79) who were receiving SGLT2i agents plus standard of care [SOC] management and compared them to (n=56) similar diabetic patients who were only on SOC management. Results The two groups were comparable regarding age, sex, type of donor, type of diabetes (T2D PTDM), post-transplant period, induction immunosuppression and use of CNI. Though improvement of HbA1c was not significantly different between the two groups, patients on SGLT2i showed better drop in HbA1c compared to the SOC group (0.7% versus 0.5% respectively). Reduction of BMI was equal between the two groups (-1.1%) and there was no significant difference in the number of blood pressure medications (average 2 drugs per patient). Kidney function was assessed by the eGFR using CKD-EPI equation and by urine albumin/creatinine ratio (ACR). The eGFR was calculated at start then at 1,3,6 and 12 months. In SGLT2i group, eGFR showed a dip at 3 months (from 66 to 63.35 ml/min) then started to improve gradually toward the end of the year and maintained at a level close to baseline (65.44 ml/min). The SOC group showed gradual drop in eGFR over the year from 65.76 to 63.19 ml/min. Urine ACR reduced in the SGLT2i group from 48.79 to 23.79 mg/mmol creatinine and increased from 42.84 to 63.16 mg/mmol creatinine in the SOC group. The incidences of graft rejection, urinary tract infection, genital infection, myocardial infarction, heart failure or cerebrovascular stroke were not different between the groups. Conclusion Use of SGLT2i in managing diabetic patients post kidney transplantation is safe and has better short-term outcomes on renal function with comparable safety compared to standard of care therapy.

Na+ -K+ -2Cl- cotransporter-1 (NKCC1) facilitates basolateral K+ and Cl- uptake, supporting their efflux across mucosal membranes of colonic epithelial cells. NKCC1 activity has also been shown to be critical for electrogenic K+ secretion induced by aldosterone, which is known to stimulate large-conductance K+ (BK) channel expression in mucosal membranes. This study was aimed to (1) identify whether aldosterone enhances NKCC1 expression specifically to support BK-mediated K+ secretion and (2) to determine whether increased NKCC1 supports electrogenic Cl- secretion in parallel to K+ secretion. Dietary Na+ depletion was used to induce secondary hyperaldosteronism in rats, or aldosterone was administered ex vivo to rat distal colonic mucosae. NKCC1-dependent electrogenic K+ or Cl- secretion was measured as a function of short circuit current (ISC ). qRT-PCR, western blot, and immunofluorescence analyses were performed using standard techniques. Aldosterone enhanced NKCC1 and BKα expression and electrogenic K+ secretion in the distal colon, which was inhibited by either serosal bumetanide (NKCC1 inhibitor) or mucosal iberiotoxin (IbTX; BK channel blocker), but not TRAM-34 (IK channel blocker). Expression of NKCC1 and BKα proteins was enhanced in crypt cells of hyper-aldosterone rats. However, neither NKCC1-dependent Cl- secretion nor CFTR (apical Cl- channel) expression was enhanced by aldosterone. We conclude that aldosterone enhances NKCC1 to support BK-mediated K+ secretion independently of Cl- secretion in the distal colon. The regulation of NKCC1 expression/K+ secretion by aldosterone may be a therapeutic target in treating gastrointestinal disorders associated with alterations in colonic K+ transport, such as colonic pseudo-obstruction, and hyperkalemia associated with renal disease.

Multiple evanescent white dot syndrome (MEWDS) is an inflammatory condition of the retina that typically presents unilaterally with multiple gray-white spots in the outer retina or retinal pigmented epithelium and granular changes within the fovea. We report a case of new-onset MEWDS in a patient closely after the initiation of empagliflozin, a sodium-glucose cotransporter inhibitor medicine for his type II diabetes mellitus.

With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.

Sodium-glucose cotransporter (SGLT) 2 inhibitors, which inhibit renal reabsorption of glucose and stimulate urinary glucose excretion, have been proposed as useful drugs for treating diabetes. This study examined the effects of the SGLT2 inhibitor ipragliflozin on blood glucose-dependent urinary glucose excretion in mice. To investigate the antihyperglycemic effects of ipragliflozin, several oral glucose tolerance tests (glucose load: 1-4 g/kg) were performed. Ipragliflozin significantly suppressed the increases in blood glucose levels concomitant with urinary glucose excretion. To investigate the risk of hypoglycemia, the effects of ipragliflozin were examined under fasting conditions in mice that prefasted for various time periods (0-24 h). Ipragliflozin significantly decreased blood glucose levels in mice that had prefasted for 0 or 6 h (non-hypoglycemic conditions), but not in mice that had prefasted for 12 h or more (hypoglycemic conditions). These ipragliflozin-induced hypoglycemic and urinary glucose excretion effects were well correlated with blood glucose levels. These results suggest that ipragliflozin-induced increases in urinary glucose excretion and accompanying hypoglycemic effects were caused by blood glucose-dependent mechanisms. Thus, ipragliflozin may be a useful antidiabetic agent that achieves ideal blood glucose control in diabetic patients with little risk of hypoglycemia.

Fumonisin B1 (FB1 ) is a common environmental mycotoxin produced by molds such as Fusarium verticillioides. The toxin poses health risks to domestic animals, including pigs, through FB1 -contaminanted feed. However, the cytotoxicity of FB1 to porcine intestines has not been fully analyzed. In the present study, the effects of FB1 on oxidative stress and nutrient transporter-associated genes of the porcine intestinal IPEC-J2 cells were explored. FB1 decreased IPEC-J2 proliferation but did not trigger reactive oxygen species (ROS) overproduction. Meanwhile, FB1 reduced the expression levels of the transporters l-type amino acid transporter-1 (y+ LAT1), solute carrier family 7 member 1 (SLC7A1), solute carrier family 1 member 5 (ASCT2), and excitatory amino acid carrier 1 (EAAC1); in addition, FB1 reduced the levels of the fatty acid transporters long-chain fatty acid transport protein 1 (FATP1) and long-chain fatty acid transport protein 4 (FATP4) as well as glucose transporters Na+ /glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2). FB1 stimulation increased the expression levels of peptide transporter peptide transporter 1 (PepT1) and metal ion transport-related gene zinc transporter 1 (ZNT1). Moreover, metal ion transporter divalent metal transporter 1 (DMT1) expression was depressed by a higher dosage of FB1 . The data indicate that FB1 results in aberrant expression of nutrient transporters in IPEC-J2 cells, thereby exerting its toxicity even though it fails to exert ROS-dependent oxidative stress.

Na+/H+ exchange factor-1 (NHERF1), a multidomain PDZ scaffolding phosphoprotein, is required for the type II sodium-dependent phosphate cotransporter (NPT2A)-mediated renal phosphate absorption. Both PDZ1 and PDZ2 domains are involved in NPT2A-dependent phosphate uptake. Though harboring identical core-binding motifs, PDZ1 and PDZ2 play entirely different roles in hormone-regulated phosphate transport. PDZ1 is required for the interaction with the C-terminal PDZ-binding sequence of NPT2A (-TRL). Remarkably, phosphocycling at Ser290 distant from PDZ1, the penultimate step for both parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) regulation, controls the association between NHERF1 and NPT2A. PDZ2 interacts with the C-terminal PDZ-recognition motif (-TRL) of G Protein-coupled Receptor Kinase 6A (GRK6A), and that promotes phosphorylation of Ser290. The compelling biological puzzle is how PDZ1 and PDZ2 with identical GYGF core-binding motifs specifically recognize distinct binding partners. Binding determinants distinct from the canonical PDZ-ligand interactions and located “outside the box” explain PDZ domain specificity. Phosphorylation of NHERF1 by diverse kinases and associated conformational changes in NHERF1 add more complexity to PDZ-binding diversity.

The nuchal organ of the embryos and neonates of the cladoceran, Daphnia magna, has been shown to be a site of Na+ influx and H+, NH4+ and Cl- efflux. This study combines the scanning-ion selective electrode technique with application of inhibitors of specific transporters to assess the mechanisms of Na+ transport across the nuchal organ. Na+ influx across the nuchal organ was inhibited both by inhibitors of the Na+/K+-ATPase (ouabain, bufalin) and by inhibitors of the vacuolar H+-ATPase (bafilomycin, N-ethylmaleimde, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, KM91104, S-nitrosoglutathione). Na+ influx was unaffected by the epithelial Na+ channel blocker benzamil, but was sensitive to ethylisopropyl amiloride and elevated external ammonium concentrations, consistent with roles for Na+/H+ and Na+/NH4+ exchangers in the apical membrane but not Na+ channels. Transport across the basolateral membrane into the hemolymph is proposed to involve the Na+/K+-ATPase and a thiazide-sensitive Na+:Cl- cotransporter. Keywords: Daphnia magna, Na+/K+-ATPase, V-ATPase, Iionoregulation, Nnuchal organ.