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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.

BACKGROUND AND OBJECTIVES: The estimated glomerular filtration rate (eGFR) is a crucial parameter in heart failure. Much less is known about the importance of tubular function. We addressed the effect of tubular maximum phosphate reabsorption capacity (TmP/GFR), a parameter of proximal tubular function, in patients with heart failure.DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We established TmP/GFR (Bijvoet formula) in 2085 patients with heart failure and studied its association with deterioration of kidney function (>25% eGFR decrease from baseline) and plasma neutrophil gelatinase-associated lipocalin (NGAL) doubling (baseline to 9 months) using logistic regression analysis and clinical outcomes using Cox proportional hazards regression. Additionally, we evaluated the effect of sodium-glucose transport protein 2 (SGLT2) inhibition by empagliflozin on tubular maximum phosphate reabsorption capacity in 78 patients with acute heart failure using analysis of covariance.RESULTS: Low TmP/GFR (CONCLUSIONS: TmP/GFR, a measure of proximal tubular function, is frequently reduced in heart failure, especially in patients with more advanced heart failure. Lower TmP/GFR is furthermore associated with future risk of plasma NGAL doubling and worse clinical outcomes, independent of glomerular function.

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.

As the main exchanger of electroneutral NaCl absorption, sodium-hydrogen exchanger isoform 3 (NHE3) circulates in the epithelial brush border (BB) and intracellular compartments in a multi-protein complex. The size of the NHE3 complex changes during rapid regulation events. Recycling regulation of NHE3 in epithelial cells can be roughly divided into three stages. First, when stimulated by Ca(2+), cGMP, and cAMP-dependent signaling pathways, NHE3 is converted from an immobile complex found at the apical microvilli (MV) into an easily internalized and mobile form that relocates to a compartment near the base of the MV. Second, NHE3 is internalized by clathrin and albumin-dependent pathways into cytoplasmic endosomal compartments, where the complex is reprocessed and reassembled. Finally, NHE3 is translocated from the recycling endosomes (REs) to the apex of epithelial cells, a process that can be stimulated by an increase in sodium-glucose cotransporter 1 (SGLT1) activity, epidermal growth factor receptor (EGFR) signaling, Ca(2+) signaling, and binding to βPix and SH3 and multiple ankyrin repeat domains 2 (Shank2) proteins. This review describes the molecular steps and protein interactions involved in the recycling movement of NHE3 from the apex of epithelial cells, into vesicles, where it is reprocessed and reassembled, and returned to its original location on the plasma membrane, where it exerts its physiological function.

Estrogen-related receptors (ERRs) are known to function in mammalian kidney as key regulators of ion transport-related genes; however, a comprehensive understanding of the physiological functions of ERRs in vertebrate body fluid ionic homeostasis is still elusive. Here, we used medaka (Oryzias melastigma), a euryhaline teleost, to investigate how ERRs are involved in ion regulation. After transferring medaka from hypertonic seawater to hypotonic freshwater (FW), the mRNA expression levels of errγ2 were highly upregulated, suggesting that Errγ2 may play a crucial role in ion uptake. In situ hybridization showed that errγ2 was specifically expressed in ionocytes, the cells responsible for Na+/Cl− transport. In normal FW, ERRγ2 morpholino knockdown caused reductions in the mRNA expression of Na+/Cl− cotransporter (Ncc), the number of Ncc ionocytes, Na+/Cl− influxes of ionocytes, and whole-body Na+/Cl− contents. In FW with low Na+ and low Cl−, the expression levels of mRNA for Na+/H+ exchanger 3 (Nhe3) and Ncc were both decreased in Errγ2 morphants. Treating embryos with DY131, an agonist of Errγ, increased the whole-body Na+/Cl− contents and ncc mRNA expression in Errγ2 morphants. As such, medaka Errγ2 may control Na+/Cl− uptake by regulating ncc and/or nhe3 mRNA expression and ionocyte number, and these regulatory actions may be subtly adjusted depending on internal and external ion concentrations. These findings not only provide new insights into the underpinning mechanism of actions of ERRs, but also enhance our understanding of their roles in body fluid ionic homeostasis for adaptation to changing environments during vertebrate evolution.

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

Astrocytes are pivotal responders to alterations of extracellular pH, primarily by regulation of their principal acid-base transporter, the membrane-bound electrogenic Na+ /bicarbonate cotransporter 1 (NBCe1). Here, we describe amammalian target of rapamycin (mTOR)-dependent and NBCe1-mediated astroglial response to extracellular acidosis. Using primary mouse cortical astrocytes, we investigated the effect of long-term extracellular metabolic acidosis on regulation of NBCe1 and elucidated the underlying molecular mechanisms by immunoblotting, biotinylation of surface proteins, intracellular H+ recording using the H+ -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, and phosphoproteomic analysis. The results showed significant increase of NBCe1-mediated recovery of intracellular pH from acidification in WT astrocytes, but not in cortical astrocytes from NBCe1-deficient mice. Acidosis-induced upregulation of NBCe1 activity was prevented following inhibition of mTOR signaling by rapamycin. Yet, during acidosis or following exposure of astrocytes to rapamycin, surface protein abundance of NBCe1 remained -unchanged. Mutational analysis in HeLa cells suggested that NBCe1 activity was dependent on phosphorylation state of Ser245 , a residue conserved in all NBCe1 variants. Moreover, phosphorylation state of Ser245 is regulated by mTOR and is inversely correlated with NBCe1 transport activity. Our results identify pSer245 as a novel regulator of NBCe1 functional expression. We propose that context-dependent and mTOR-mediated multisite phosphorylation of serine residues of NBCe1 is likely to be a potent mechanism contributing to the response of astrocytes to acid/base challenges during pathophysiological conditions.

A number of well organised randomised control trials have demonstrated benefits of sodium-glucose cotransporter 2 Inhibitors and glucagon-like peptide 1 receptor agonists in reducing major adverse cardiac events. Secondary endpoints for SGLT-2is have also shown improvement in outcomes for those with heart failure with reduced ejection fraction and chronic kidney disease with albuminuria. These therapeutic advantages enable risk stratification and for treatment to be individualised depending on patient baseline characteristics. This article discusses the place of different therapeutic agents in the treatment of type 2 diabetes and describes why we should adopt a holistic approach in managing the cardio-renal risk associated with type 2 diabetes in light of the current best practice evidence.

An acute reduction in plasma osmolality causes rapid uptake of water by astrocytes but not by neurons, whereas both cell types swell as a consequence of lost blood flow (ischemia). Either hypoosmolality or ischemia can displace the brain downwards, potentially causing death. However, these disorders are fundamentally different at the cellular level. Astrocytes osmotically swell or shrink because they express functional water channels (aquaporins), whereas neurons lack functional aquaporins and thus maintain their volume. Yet both neurons and astrocytes immediately swell when blood flow to the brain is compromised (cytotoxic edema) as following stroke onset, sudden cardiac arrest, or traumatic brain injury. In each situation, neuronal swelling is the direct result of spreading depolarization (SD) generated when the ATP-dependent sodium/potassium ATPase (the Na+/K+ pump) is compromised. The simple, and incorrect, textbook explanation for neuronal swelling is that increased Na+ influx passively draws Cl− into the cell, with water following by osmosis via some unknown conduit. We first review the strong evidence that mammalian neurons resist volume change during acute osmotic stress. We then contrast this with their dramatic swelling during ischemia. Counter-intuitively, recent research argues that ischemic swelling of neurons is non-osmotic, involving ion/water cotransporters as well as at least one known amino acid water pump. While incompletely understood, these mechanisms argue against the dogma that neuronal swelling involves water uptake driven by an osmotic gradient with aquaporins as the conduit. Promoting clinical recovery from neuronal cytotoxic edema evoked by spreading depolarizations requires a far better understanding of molecular water pumps and ion/water cotransporters that act to rebalance water shifts during brain ischemia.

The divalent anion sodium symporter (DASS) family contains both sodium-driven anion co-transporters and anion/anion exchangers. The family belongs to a broader Ion Transporter Superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets. We recently determined multiple structures of the human sodium-dependent citrate transporter (NaCT) and the succinate/dicarboxylate transporter from Lactobacillus acidophilus (LaINDY). Structures of both proteins show high degrees of structural similarity to the previously-determined VcINDY fold. Conservation between these DASS protein structures and those from the AbgT familiy indicates that the VcINDY fold represents the overall protein structure for the entire ITS superfamily. The new structures of NaCT and LaINDY are captured in the inward- or outward-facing conformations, respectively. The domain arrangements in these structures agree with a rigid-body elevator-type transport mechanism for substrate translocation across the membrane. Two separate NaCT structures in complex with a substrate or an inhibitor allowed us to explain the inhibition mechanism and propose a detailed classification scheme for grouping disease-causing mutations in the human protein. Structural understanding of multiple kinetic states of DASS proteins is a first step towards the detailed characterization of their entire transport cycle.

Giant clams live in symbiosis with phototrophic dinoflagellates. They need to increase the uptake of inorganic carbon (Ci) from the ambient seawater to support light-enhanced shell formation in the host and photosynthesis in the symbionts during illumination. The ctenidium is the major site of light-enhanced Ci absorption in the fluted giant clam, Tridacna squamosa. Catalyzed by dual-domain carbonic anhydrase, exogenous HCO3− is dehydrated to CO2, which permeates the apical membrane of the ctenidial epithelium and is rehydrated back to HCO3− in the cytoplasm. However, the molecular mechanism that transports cytoplasmic HCO3− through the basolateral membrane to the hemolymph has not been elucidated. We have obtained from the ctenidium of T. squamosa the complete cDNA coding sequence of a homolog of electrogenic Na+–HCO3− cotransporter 1 (NBCe1-like), which comprised 3450 bp, encoding a protein (NBCe1-like) of 1142 amino acids and 128.9 kDa. NBCe1-like had a basolateral localization in epithelial cells covering the ctenidial filament and those surrounding the tertiary water channels. Light exposure led to significant increases in the transcript and protein levels of NBCe1-like/NBCe1-like in the ctenidium of T. squamosa, indicating that NBCe1-like could be involved in the increased transport of cytoplasmic HCO3− across the basolateral membrane into the hemolymph during illumination. Additionally, NBCe1-like might also participate in light-enhanced NO3− absorption in T. squamosa, due to the replacement of aspartate (residue 585) with glutamine. Exogenous NO3− could be absorbed by the ctenidial epithelial cells through the apical H+:NO3− cotransporter (SIALIN) and then transported through the basolateral NBCe1-like to the hemolymph.

The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline-alanine-rich kinase (SPAK) are physiological substrates of the with-no-lysine (WNK) kinase. They are the master regulators of cation Cl- cotransporters that could be targeted for discovering novel antihypertensive agents. Both kinases have a conserved carboxy-terminal (CCT) domain that recognizes a unique peptide motif (Arg-Phe-Xaa-Val) present in their upstream kinases and downstream substrates. Here, we have combined molecular docking with molecular dynamics simulations and free-energy calculations to identify potential inhibitors that can bind to the allosteric pocket of the OSR1-CCT domain and impede its interaction with the WNK peptide. Our study revealed that STOCK1S-14279 and Closantel bound strongly to the allosteric pocket of OSR1 and displaced the WNK peptide from the primary pocket of OSR1. We showed that primarily Arg1004 and Gln1006 of the WNK4-peptide motif were involved in strong H-bond interactions with Glu453 and Arg451 of OSR1. Besides, our study revealed that atoms of Arg1004 were solvent-exposed in cases of STOCK1S-14279 and Closantel, implying that the WNK4 peptide was moved out of the pocket. Overall, the predicted potential inhibitors altogether abolish the OSR1-WNK4-peptide interaction, suggesting their potency as a prospective allosteric inhibitor against OSR1.

Diabetes is a known risk factor for the development of cardiovascular and chronic kidney disease. Sodium–glucose cotransporter 2 inhibitors are glucose-lowering medications that have been demonstrated to improve clinical cardiovascular and renal outcomes. This article reviews recent large clinical trials involving these medications and explains their impact on type 2 diabetes treatment guidelines.

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.

KEY POINTS GABA depolarized sural nerve axons and increased the electrical excitability of C-fibres via GABAA receptor. Axonal excitability responses to GABA increased monotonically with the rate of action potential firing. Action potential activity in unmyelinated C-fibres is coupled to Na-K-Cl cotransporter type 1 (NKCC1) loading of axonal chloride. Activation of axonal GABAA receptor stabilized C-fibre excitability during prolonged low frequency (2.5 Hz) firing. NKCC1 maintains intra-axonal chloride to provide feed-forward stabilization of C-fibre excitability and thus support sustained firing. ABSTRACT GABAA receptor (GABAA R)-mediated depolarization of dorsal root ganglia (DRG) axonal projections in the spinal dorsal horn is implicated in pre-synaptic inhibition. Inhibition, in this case, is predicated on an elevated intra-axonal chloride concentration and a depolarizing GABA response. In the present study, we report that the peripheral axons of DRG neurons are also depolarized by GABA and this results in an increase in the electrical excitability of unmyelinated C-fibre axons. GABAA R agonists increased axonal excitability, whereas GABA excitability responses were blocked by GABAA R antagonists and were absent in mice lacking the GABAA R β3 subunit selectively in DRG neurons (AdvillinCre or snsCre ). Under control conditions, excitability responses to GABA became larger at higher rates of electrical stimulation (0.5-2.5 Hz). However, during Na-K-Cl cotransporter type 1 (NKCC1) blockade, the electrical stimulation rate did not affect GABA response size, suggesting that NKCC1 regulation of axonal chloride is coupled to action potential firing. To examine this, activity-dependent conduction velocity slowing (activity-dependent slowing; ADS) was used to quantify C-fibre excitability loss during a 2.5 Hz challenge. ADS was reduced by GABAA R agonists and exacerbated by either GABAA R antagonists, β3 deletion or NKCC1 blockade. This illustrates that activation of GABAA R stabilizes C-fibre excitability during sustained firing. We posit that NKCC1 acts in a feed-forward manner to maintain an elevated intra-axonal chloride in C-fibres during ongoing firing. The resulting chloride gradient can be utilized by GABAA R to stabilize axonal excitability. The data imply that therapeutic strategies targeting axonal chloride regulation at peripheral loci of pain and itch may curtail aberrant firing in C-fibres.

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.

The secondary-active Na-K-Cl cotransporter 1 (NKCC1), member of the cation-chloride cotransporter (CCC) family, ensures the electroneutral movement of Cl-, Na+, and K+ ions across cellular membranes. NKCC1 regulates Cl- homeostasis and cell volume, handling a pivotal role in transepithelial water transport and neuronal excitability. Aberrant NKCC1 transport is hence implicated in a variety of human diseases (hypertension, renal disorders, neuropathies, and cancer). Building on the newly resolved NKCC1 cryo-EM structure, all-atom enhanced sampling simulations unprecedentedly unlock the mechanism of NKCC1-mediated ion transport, assessing the order and the molecular basis of its interdependent ion translocation. Our outcomes strikingly advance the understanding of the physiological mechanism of CCCs and disclose a key role of CCC-conserved asparagine residues, whose side-chain promiscuity ensures the transport of both negatively and positively charged ions along the same translocation route. This study sets a conceptual basis to devise NKCC-selective inhibitors to treat diseases linked to Cl- dishomeostasis.

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.

The renal nephron consists of a series of distinct cell types that function in concert to maintain fluid and electrolyte balance and blood pressure. The renin-angiotensin system (RAS) is central to Na(+) and volume balance. We aimed to determine how loss of angiotensin II signaling in the proximal tubule (PT), which reabsorbs the bulk of filtered Na(+) and volume, impacts solute transport throughout the nephron. We hypothesized that PT renin-angiotensin system disruption would not only depress PT Na(+) transporters but also impact downstream Na(+) transporters. Using a mouse model in which the angiotensin type 1a receptor (AT(1a)R) is deleted specifically within the PT (AT(1a)R PTKO), we profiled the abundance of Na(+) transporters, channels, and claudins along the nephron. Absence of PT AT(1a)R signaling was associated with lower abundance of PT transporters (Na(+)/H(+) exchanger isoform 3, electrogenic Na(+)-bicarbonate cotransporter 1, and claudin 2) as well as lower abundance of downstream transporters (total and phosphorylated Na(+)-K(+)-2Cl(−) cotransporter, medullary Na(+)-K(+)-ATPase, phosphorylated NaCl cotransporter, and claudin 7) versus controls. However, transport activities of Na(+)-K(+)-2Cl(−) cotransporter and NaCl cotransporter (assessed with diuretics) were similar between groups in order to maintain electrolyte balance. Together, these results demonstrate the primary impact of angiotensin II regulation on Na(+) reabsorption in the PT at baseline and the associated influence on downstream Na(+) transporters, highlighting the ability of the nephron to integrate Na(+) transport along the nephron to maintain homeostasis. NEW & NOTEWORTHY Our study defines a novel role for proximal tubule angiotensin receptors in regulating the abundance of Na(+) transporters throughout the nephron, thereby contributing to the integrated control of fluid balance in vivo.

High sodium (HS) intake inhibited epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron and Na+-Cl- cotransporter (NCC) by suppressing basolateral Kir4.1/Kir5.1 in the distal convoluted tubule (DCT), thereby increasing renal Na+ excretion but not affecting K+ excretion. The aim of the present study was to explore whether deletion of Kir5.1 compromises the inhibitory effect of HS on NCC expression/activity and renal K+ excretion. Patch-clamp experiments demonstrated that HS failed to inhibit DCT basolateral K+ channels and did not depolarize K+ current reversal potential of the DCT in Kir5.1 knockout (KO) mice. Moreover, deletion of Kir5.1 not only increased the expression of Kir4.1, phospho-NCC, and total NCC but also abolished the inhibitory effect of HS on the expression of Kir4.1, phospho-NCC, and total NCC and thiazide-induced natriuresis. Also, low sodium-induced stimulation of NCC expression/activity and basolateral K+ channels in the DCT were absent in Kir5.1 KO mice. Deletion of Kir5.1 decreased ENaC currents in the late DCT, and HS further inhibited ENaC activity in Kir5.1 KO mice. Finally, measurement of the basal renal K+ excretion rate with the modified renal clearance method demonstrated that long-term HS inhibited the renal K+ excretion rate and steadily increased plasma K+ levels in Kir5.1 KO mice but not in wild-type mice. We conclude that Kir5.1 plays an important role in mediating the effect of HS intake on basolateral K+ channels in the DCT and NCC activity/expression. Kir5.1 is involved in maintaining renal ability of K+ excretion during HS intake. NEW & NOTEWORTHY Kir5.1 plays an important role in mediating the effect of high sodium intake on basolateral K+ channels in the distal convoluted tubule and Na+-Cl- cotransporter activity/expression.

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.

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.

Neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) regulates the expression of Kir4.1, thiazide-sensitive NaCl cotransporter (NCC), and epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN), and Nedd4-2 deletion causes salt-sensitive hypertension. We now examined whether Nedd4-2 deletion compromises the effect of high-salt (HS) diet on Kir4.1, NCC, ENaC, and renal K(+) excretion. Immunoblot analysis showed that HS diet decreased the expression of Kir4.1, Ca(2+)-activated large-conductance K(+) channel subunit-α (BKα), ENaCβ, ENaCγ, total NCC, and phospho-NCC (at Thr(53)) in floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l(fl/fl)) mice, whereas these effects were absent in kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. Renal clearance experiments also demonstrated that Nedd4-2 deletion abolished the inhibitory effect of HS diet on hydrochlorothiazide-induced natriuresis. Patch-clamp experiments showed that neither HS diet nor low-salt diet had an effect on Kir4.1/Kir5.1 currents of the distal convoluted tubule in Nedd4-2-deficient mice, whereas we confirmed that HS diet inhibited and low-salt diet increased Kir4.1/Kir5.1 activity in Nedd4l(flox/flox) mice. Nedd4-2 deletion increased ENaC currents in the ASDN, and this increase was more robust in the cortical collecting duct than in the distal convoluted tubule. Also, HS-induced inhibition of ENaC currents in the ASDN was absent in Nedd4-2-deficient mice. Renal clearance experiments showed that HS intake for 2 wk increased the basal level of renal K(+) excretion and caused hypokalemia in Ks-Nedd4-2-KO mice but not in Nedd4l(flox/flox) mice. In contrast, plasma Na(+) concentrations were similar in Nedd4l(flox/flox) and Ks-Nedd4-2 KO mice on HS diet. We conclude that Nedd4-2 plays an important role in mediating the inhibitory effect of HS diet on Kir4.1, ENaC, and NCC and is essential for maintaining normal renal K(+) excretion and plasma K(+) ranges during long-term HS diet. NEW & NOTEWORTHY The present study suggests that Nedd4-2 is involved in mediating the inhibitory effect of high salt (HS) diet on Kir4.1/kir5.1 in the distal convoluted tubule, NaCl cotransporter function, and epithelial Na(+) channel activity and that Nedd4-2 plays an essential role in maintaining K(+) homeostasis in response to a long-term HS diet. This suggests the possibility that HS intake could lead to hypokalemia in subjects lacking proper Nedd4-2 E3 ubiquitin ligase activity in aldosterone-sensitive distal nephron.

Hypertension is the largest risk factor for cardiovascular disease, the leading cause of mortality worldwide. As blood pressure regulation is influenced by multiple physiological systems, hypertension cannot be attributed to a single identifiable etiology. Three decades of research into Mendelian forms of hypertension implicated alterations in the renal tubular sodium handling, particularly the distal convoluted tubule (DCT)-native, thiazide-sensitive Na–Cl cotransporter (NCC). Altered functions of the NCC have shown to have profound effects on blood pressure regulation as illustrated by the over activation and inactivation of the NCC in Gordon's and Gitelman syndromes respectively. Substantial progress has uncovered multiple factors that affect the expression and activity of the NCC. In particular, NCC activity is controlled by phosphorylation/dephosphorylation, and NCC expression is facilitated by glycosylation and negatively regulated by ubiquitination. Studies have even found parvalbumin to be an unexpected regulator of the NCC. In recent years, there have been considerable advances in our understanding of NCC control mechanisms, particularly via the pathway containing the with-no-lysine [K] (WNK) and its downstream target kinases, SPS/Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress responsive 1 (OSR1), which has led to the discovery of novel inhibitory molecules. This review summarizes the currently reported regulatory mechanisms of the NCC and discusses their potential as therapeutic targets for treating hypertension., Graphical abstract This review proposes integrated model of NCC regulation, and summarizes therapeutic strategies to target the NCC via its regulatory pathway.Image 1

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.

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.

Hyperglycemia is a key therapeutic focus in the management of patients with type 2 diabetes (T2D) mellitus. The various therapeutic classes of antidiabetic drugs presently existing in the market are not sufficiently effective in maintaining long-term glycemic control in most of the diabetic patients, even when used in combination. The undesirable adverse effects of these drugs, such as hypoglycemia, weight gain, and hepatic and renal toxicity, have escalated the demand for the discovery of new and safer antidiabetic drugs. The progressive nature of T2D requires practitioners to periodically evaluate patients and intensify glucose-lowering treatment once glycemic targets are not attained. Sodium-glucose cotransporter 2 inhibitors (SGLT2-is) are the new class of antidiabetic medications that are approved (2013) by the Food and Drug Administration recently for treating diabetes. These inhibitors block the SGLT2 protein involved in glucose reabsorption from the proximal renal tubule resulting in escalated glucose excretion and lower blood glucose levels. These inhibitors exhibit favorable effects beyond glucose control, such as consistent body weight, blood pressure, and serum uric acid reductions. This review highlighted the brief updates of SGLT2-i, their benefits, and adverse effects.

With no lysine kinase-4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that causes familial hyperkalemic hypertension. This disease is mainly driven by increased downstream activation of the Ste20/SPS1-related proline-alanine-rich kinase/oxidative stress responsive kinase-1-NCC pathway, which increases salt reabsorption in the distal convoluted tubule and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

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.

Children are more likely to develop epileptic seizures (ictal discharges lasting tens of seconds) than adults. A higher predisposition of the juvenile brain to the generation of epileptiform activity is thought to be due to a prevalence of inhibition over excitation at the early stage of brain development. However, the molecular and physiological mechanisms underlying these age-related differences are yet to be clarified. We compared the ictal activity induced by a convulsant 4-aminopyridine (4-AP) in the horizontal slices of the entorhinal cortex and hippocampus of 3- and 8-week-old Wistar rats. In 3-week-old rats, the ictal discharge was always preceded by a detectable preictal activity, as manifested in one or several 3–4-s GABA-glutamate events, whereas in 8-week-old rats, such events were typically absent or very rare (no more than one occasional short event). The ictal activity resistance to external exposures was also age-dependent. In 8-week-old rats, by contrast to 3-week-old animals, ictal discharge generation in the entorhinal cortex was blocked completely and replaced by 0.2–0.3 Hz interictal activity (simultaneous 1–3-s burst discharges) by a partial blockade of KCC2 cotransporter or Na+–K+-pump, as well as by low-frequency electric stimulation. Thus, our data indicate that ictal discharges in the immature (3-week-old) brain are more resistant to external exposures than in the brain of adult rats. Interictal and ictal epileptiform activities are antagonistic in 8-week-old animals. In contrast, the appearance of interictal activity interrupts the generation of ictal discharges completely. It can therefore be considered as one of the putative antiepileptic mechanisms in the mature rat brain.

Our aim was to use theta coherence as a quantitative trait to investigate the relation of the polymorphisms in NKCC1 (rs3087889) and KCC2 (rs9074) channel protein genes to autistic traits (AQ) in neurotypicals. Coherence values for candidate connection regions were calculated from eyes-closed resting EEGs in two independent groups. Hypersynchrony within the right anterior region was related to AQ in both groups (p

Aim: Sodium–glucose cotransporter 2 inhibitors (SGLT2is) provide renal protection in patients with type 2 diabetes mellitus (T2DM). The aim of this study was to elucidate the renal effects of long-...

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.

Seizures invite seizures. At the initial stage of epilepsy, seizures intensify with each episode; however, the mechanisms underlying this exacerbation remain to be solved. Astrocytes have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K+clearance relies on the K+uptake by astrocytes and the subsequent diffusion of K+through the astrocyte network. When astrocytes become uncoupled, K+clearance becomes hindered. Accumulation of extracellular K+leads to hyperexcitability of neurons. Here, using acute hippocampal slices from mice, we uncovered that brief periods of epileptiform activity result in gap junction uncoupling. In slices that experienced short-term epileptiform activity, extracellular K+transients in response to glutamate became prolonged. Na+imaging with a fluorescent indicator indicated that intercellular diffusion of small cations in the astrocytic syncytium via gap junctions became rapidly restricted after epileptiform activity. Using a transgenic mouse with astrocyte-specific expression of a pH sensor (Lck-E2GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na+/HCO3–cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification bothin vitroandin vivo. Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.SIGNIFICANCE STATEMENTWe aimed to understand the mechanisms underlying the plastic change of forebrain circuits associated with the intensification of epilepsy. Here, we demonstrate that first-time exposure to only brief periods of epileptiform activity results in acute disturbance of the intercellular astrocyte network formed by gap junctions in hippocampal tissue slices from mice. Moreover, rapid clearance of K+from the extracellular space was impaired. Epileptiform activity activated inward Na+/HCO3–cotransport in astrocytes by cell depolarization, resulting in their alkalization. Our data suggest that alkaline pH shifts in astrocytes lead to gap junction uncoupling, hampering K+clearance, and thereby to exacerbation of epilepsy. Pharmacological intervention could become a promising new strategy to dampen neuronal hyperexcitability and epileptogenesis.

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.

BACKGROUND: The electroneutral Na +/HCO 3 - cotransporter NBCn1 (Slc4a7) is expressed in basolateral membranes of renal medullary thick ascending limbs (mTALs). However, direct evidence that NBCn1 contributes to acid-base handling in mTALs, urinary net acid excretion, and systemic acid-base homeostasis has been lacking. METHODS: Metabolic acidosis was induced in wild-type and NBCn1 knockout mice. Fluorescence-based intracellular pH recordings were performed and NH 4 + transport measured in isolated perfused mTALs. Quantitative RT-PCR and immunoblotting were used to evaluate NBCn1 expression. Tissue [NH 4 +] was measured in renal biopsies, NH 4 + excretion and titratable acid quantified in spot urine, and arterial blood gasses evaluated in normoventilated mice. RESULTS: Basolateral Na +/HCO 3 - cotransport activity was similar in isolated perfused mTALs from wild-type and NBCn1 knockout mice under control conditions. During metabolic acidosis, basolateral Na +/HCO 3 - cotransport activity increased four-fold in mTALs from wild-type mice, but remained unchanged in mTALs from NBCn1 knockout mice. Correspondingly, NBCn1 protein expression in wild-type mice increased ten-fold in the inner stripe of renal outer medulla during metabolic acidosis. During systemic acid loading, knockout of NBCn1 inhibited the net NH 4 + reabsorption across mTALs by approximately 60%, abolished the renal corticomedullary NH 4 + gradient, reduced the capacity for urinary NH 4 + excretion by approximately 50%, and delayed recovery of arterial blood pH and standard [HCO 3 -] from their initial decline. CONCLUSIONS: During metabolic acidosis, NBCn1 is required for the upregulated basolateral HCO 3 - uptake and transepithelial NH 4 + reabsorption in mTALs, renal medullary NH 4 + accumulation, urinary NH 4 + excretion, and early recovery of arterial blood pH and standard [HCO 3 -]. These findings support that NBCn1 facilitates urinary net acid excretion by neutralizing intracellular H + released during NH 4 + reabsorption across mTALs.

Unicellular algae have evolved to express many forms of high-affinity phosphate transporters, and homologs of these proteins are broadly distributed in yeast, fungi, higher plants, and vertebrates. In this report, an effort has been made to characterize such a transporter gene, StPHO, in the marine diatom Skeletonema tropicum. The primers used for polymerase chain reaction were designed by referring to a homologous gene in a prasinophyte, and the full-length (1692 bp) cDNA of StPHO was then cloned and sequenced. Sequence alignments and secondary structure prediction indicated that StPHO is a gene that encodes a type III Na+/Pi cotransporter (SLC20 family). To study the function of StPHO, specific concentrations of inorganic phosphate (Pi) were used to alter the physiological status of S. tropicum. In each treatment, samples were collected for the measurements of StPHO mRNA, [PO4 3−], cell abundance, the maximal photochemical efficiency of photosystem II (F v /F m ), and alkaline phosphatase activity (APA). The results indicated that the ambient [PO4 3−] strongly affected the population growth and related physiological parameters of S. tropicum. The transcription of StPHO was fully repressed when the [PO4 3−] was greater than 1 μM but increased approximately 100-fold when the ambient [PO4 3−] decreased to 0.02 μM. Within this [PO4 3−] range, the regression equations are Y = −0.6644X + 0.9034 and Y = −0.5908X + 0.8054 for Pi-starved and Pi-limited treatments, respectively. This trend of gene expression suggested that StPHO plays an important role in the uptake of [PO4 3−], and StPHO may serve as a useful molecular biomarker for Pi-stressed diatom populations in marine ecosystems.

In this study, we show that the electrogenic Na+/HCO3− cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

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.

Objective . The role of Cl – -transport in ATP-dependent regulation of contractile activity of rat pulmonary artery (PA) smooth muscle cells was studied. Design and methods . The study was performed on endotheliumdenuded ring segments of the PA of male Wistar rats. Mechanical tension was measured using organ bath technique. Contractions of the PA segments were induced by high-potassium solution (30 mM KCl), hyposmotic solution (40 mM NaCl), as well as restoration of the medium osmolarity (120 mM NaCl) after incubation in hyposmotic solution. Inhibitor of Na + , K + , 2Cl – cotransport (NKCC) bumetanide (10 μM, 30 minutes preincubation), nonselective Cl – -channel blocker SITS (100 μM) and Ca2 + -activated Cl – channels blocker niflumic acid (NA, 10 μM) were used to modulate the Cl – -transport. Results . ATP (10–500 μM) did not affect vascular tone of PA segments incubated in Krebs solution (120 mM NaCl), while 500 and 1000 μM ATP led to the development of transient contractions, the amplitude of which decreased in the presence of bumetanide, SITS and NA. Incubation of PA segments in a hyposmotic medium (40 mM NaCl) caused the development of transient contraction. Subsequent recovery of the medium osmolarity (120 mM NaCl) induced another transient contraction — isosmotic striction. ATP (500 μM) eliminated the relaxation phase of hyposmotic striction, and completely suppressed the development of isosmotic striction. Bumetanide did not affect the action of ATP during isosmotic striction, but restored the relaxation phase of hyposmotic striction. SITS and NA eliminated the effect of ATP on hypo- and isosmotic striction. Conclusions . The constrictive effect of ATP on the smooth muscle cells of the PA is associated with the activation of mechanisms of transmembrane Cl – -redistribution, in which NKCC and Ca2 + -activated Cl – channels are involved.