Interplay between DISC1 and GABA signaling regulates neurogenesis in mice and risk for schizophrenia.

How extrinsic stimuli and intrinsic factors interact to regulate continuous neurogenesis in the postnatal mammalian brain is unknown. Here we show that regulation of dendritic development of newborn neurons by Disrupted-in-Schizophrenia 1 (DISC1) during adult hippocampal neurogenesis requires neurotransmitter GABA-induced, NKCC1-dependent depolarization through a convergence onto the AKT-mTOR pathway. In contrast, DISC1 fails to modulate early-postnatal hippocampal neurogenesis when conversion of GABA-induced depolarization to hyperpolarization is accelerated. Extending the period of GABA-induced depolarization or maternal deprivation stress restores DISC1-dependent dendritic regulation through mTOR pathway during early-postnatal hippocampal neurogenesis. Furthermore, DISC1 and NKCC1 interact epistatically to affect risk for schizophrenia in two independent case control studies. Our study uncovers an interplay between intrinsic DISC1 and extrinsic GABA signaling, two schizophrenia susceptibility pathways, in controlling neurogenesis and suggests critical roles of developmental tempo and experience in manifesting the impact of susceptibility genes on neuronal development and risk for mental disorders.

The biogenesis of potassium transporters: implications of disease-associated mutations.

The concentration of intracellular and extracellular potassium is tightly regulated due to the action of various ion transporters, channels, and pumps, which reside primarily in the kidney. Yet, potassium transporters and cotransporters play vital roles in all organs and cell types. Perhaps not surprisingly, defects in the biogenesis, function, and/or regulation of these proteins are linked to range of catastrophic human diseases, but to date, few drugs have been approved to treat these maladies. In this review, we discuss the structure, function, and activity of a group of potassium-chloride cotransporters, the KCCs, as well as the related sodium-potassium-chloride cotransporters, the NKCCs. Diseases associated with each of the four KCCs and two NKCCs are also discussed. Particular emphasis is placed on how these complex membrane proteins fold and mature in the endoplasmic reticulum, how non-native forms of the cotransporters are destroyed in the cell, and which cellular factors oversee their maturation and transport to the cell surface. When known, we also outline how the levels and activities of each cotransporter are regulated. Open questions in the field and avenues for future investigations are further outlined.

SIK3 and Wnk converge on Fray to regulate glial K+ buffering and seizure susceptibility.

Glial cells play a critical role in maintaining homeostatic ion concentration gradients. Salt-inducible kinase 3 (SIK3) regulates a gene expression program that controls K+ buffering in glia, and upregulation of this pathway suppresses seizure behavior in the eag, Shaker hyperexcitability mutant. Here we show that boosting the glial SIK3 K+ buffering pathway suppresses seizures in three additional molecularly diverse hyperexcitable mutants, highlighting the therapeutic potential of upregulating glial K+ buffering. We then explore additional mechanisms regulating glial K+ buffering. Fray, a transcriptional target of the SIK3 K+ buffering program, is a kinase that promotes K+ uptake by activating the Na+/K+/Cl- co-transporter, Ncc69. We show that the Wnk kinase phosphorylates Fray in Drosophila glia and that this activity is required to promote K+ buffering. This identifies Fray as a convergence point between the SIK3-dependent transcriptional program and Wnk-dependent post-translational regulation. Bypassing both regulatory mechanisms via overexpression of a constitutively active Fray in glia is sufficient to robustly suppress seizure behavior in multiple Drosophila models of hyperexcitability. Finally, we identify cortex glia as a critical cell type for regulation of seizure susceptibility, as boosting K+ buffering via expression of activated Fray exclusively in these cells is sufficient to suppress seizure behavior. These findings highlight Fray as a key convergence point for distinct K+ buffering regulatory mechanisms and cortex glia as an important locus for control of neuronal excitability.

Intraventricular haemorrhage in premature infants: the role of immature neuronal salt and water transport.

Intraventricular haemorrhage is a common complication of premature birth. Survivors are often left with cerebral palsy, intellectual disability and/or hydrocephalus. Animal models suggest that brain tissue shrinkage, with subsequent vascular stretch and tear, is an important step in the pathophysiology, but the cause of this shrinkage is unknown. Clinical risk factors for intraventricular haemorrhage are biomarkers of hypoxic-ischaemic stress, which causes mature neurons to swell. However, immature neuronal volume might shift in the opposite direction in these conditions. This is because immature neurons express the chloride, salt and water transporter NKCC1, which subserves regulatory volume increases in non-neural cells, whereas mature neurons express KCC2, which subserves regulatory volume decreases. When hypoxic-ischaemic conditions reduce active ion transport and increase the cytoplasmic membrane permeability, the effects of these transporters are diminished. Consequentially, mature neurons swell (cytotoxic oedema), whereas immature neurons might shrink. After hypoxic-ischaemic stress, in vivo and in vitro multi-photon imaging of perinatal transgenic mice demonstrated shrinkage of viable immature neurons, bulk tissue shrinkage and blood vessel displacement. Neuronal shrinkage was correlated with age-dependent membrane salt and water transporter expression using immunohistochemistry. Shrinkage of immature neurons was prevented by prior genetic or pharmacological inhibition of NKCC1 transport. These findings open new avenues of investigation for the detection of acute brain injury by neuroimaging, in addition to prevention of neuronal shrinkage and the ensuing intraventricular haemorrhage, in premature infants.

Biallelic loss-of-function variants of SLC12A9 cause lysosome dysfunction and a syndromic neurodevelopmental disorder.

PURPOSE: Pathogenic variants of FIG4 generate enlarged lysosomes and neurological and developmental disorders. To identify additional genes regulating lysosomal volume, we carried out a genome-wide activation screen to detect suppression of enlarged lysosomes in FIG4-/- cells. METHODS: The CRISPR-a gene activation screen utilized sgRNAs from the promoters of protein-coding genes. Fluorescence-activated cell sorting separated cells with correction of the enlarged lysosomes from uncorrected cells. Patient variants of SLC12A9 were identified by exome or genome sequencing and studied by segregation analysis and clinical characterization. RESULTS: Overexpression of SLC12A9, a solute co-transporter, corrected lysosomal swelling in FIG4-/- cells. SLC12A9 (NP_064631.2) colocalized with LAMP2 at the lysosome membrane. Biallelic variants of SLC12A9 were identified in 3 unrelated probands with neurodevelopmental disorders. Common features included intellectual disability, skeletal and brain structural abnormalities, congenital heart defects, and hypopigmented hair. Patient 1 was homozygous for nonsense variant p.(Arg615∗), patient 2 was compound heterozygous for p.(Ser109Lysfs∗20) and a large deletion, and proband 3 was compound heterozygous for p.(Glu290Glyfs∗36) and p.(Asn552Lys). Fibroblasts from proband 1 contained enlarged lysosomes that were corrected by wild-type SLC12A9 cDNA. Patient variant p.(Asn552Lys) failed to correct the lysosomal defect. CONCLUSION: Impaired function of SLC12A9 results in enlarged lysosomes and a recessive disorder with a recognizable neurodevelopmental phenotype.

Renal CD81 interacts with sodium potassium 2 chloride cotransporter and sodium chloride cotransporter in rats with lipopolysaccharide-induced preeclampsia.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)-induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS-PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS-PE than controls on GD14. On GD18, LPS-PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co-immunoprecipitated with NKCC2 or NCC in kidney homogenates and co-immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS-PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co-immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS-induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS-PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.

Pharmacology of Compounds Targeting Cation-Chloride Cotransporter Physiology.

Transporters of the solute carrier family 12 (SLC12) carry inorganic cations such as Na+ and/or K+ alongside Cl across the plasma membrane of cells. These tightly coupled, electroneutral, transporters are expressed in almost all tissues/organs in the body where they fulfil many critical functions. The family includes two key transporters participating in salt reabsorption in the kidney: the Na-K-2Cl cotransporter-2 (NKCC2), expressed in the loop of Henle, and the Na-Cl cotransporter (NCC), expressed in the distal convoluted tubule. NCC and NKCC2 are the targets of thiazides and "loop" diuretics, respectively, drugs that are widely used in clinical medicine to treat hypertension and edema. Bumetanide, in addition to its effect as a loop diuretic, has recently received increasing attention as a possible therapeutic agent for neurodevelopmental disorders. This chapter also describes how over the past two decades, the pharmacology of Na+ independent transporters has expanded significantly to provide novel tools for research. This work has indeed led to the identification of compounds that are 100-fold to 1000-fold more potent than furosemide, the first described inhibitor of K-Cl cotransport, and identified compounds that possibly directly stimulate the function of the K-Cl cotransporter. Finally, the recent cryo-electron microscopy revolution has begun providing answers as to where and how pharmacological agents bind to and affect the function of the transporters.

Function and regulation of the insect NaCCC2 sodium transport proteins.

NaCCC2 transport proteins, including those from Drosophila melanogaster (Ncc83) and Aedes aegypti (aeCCC2), are an insect-specific clade with sequence similarity to Na+-K+-2Cl- cotransporters. Whereas the Na+-K+-2Cl- cotransporters and other cation-chloride cotransporters are electroneutral, recent work indicates that Ncc83 and aeCCC2 carry charge across membranes. Here, we further characterize the regulation and transport properties of Ncc83 and aeCCC2 expressed in Xenopus oocytes. In cation uptake experiments, Li+ was used as a tracer for Na+ and Rb+ was used as a tracer for K+. Li+ uptake of oocytes expressing either aeCCC2 or Ncc83 was greater than uptake in water-injected controls, activated by hypotonic swelling, and not inhibited by ouabain or ethyl cinnamate. Rb+ uptake of oocytes expressing either aeCCC2 or Ncc83 was not different than water injected controls. In oocytes expressing either aeCCC2 or Ncc83, Li+ uptake plateaued with increasing Li+ concentrations, with apparent Km values in the range of 10 to 20 mM. Following exposure to ouabain, intracellular [Na+] was greater in oocytes expressing aeCCC2 than in controls. Elevating intracellular cAMP (via 8-bromo-cAMP) in Ncc83 oocytes significantly stimulated both Li+ uptake and membrane conductances. Elevating intracellular cAMP in aeCCC2 oocytes did not affect Li+ uptake, but stimulated membrane conductances. Overall, these results confirm that the NaCCC2s resemble other cation-chloride cotransporters in their regulation and some transport properties. However, unlike other cation-chloride cotransporters, they carry charge across membranes.

NKCC1 in human diseases: is the SLC12A2 gene haploinsufficient?

Mutations in the SLC12A2 gene, which encodes the Na-K-2Cl cotransporter-1 (NKCC1), are linked to various conditions such as neurodevelopmental deficits, deafness, and fluid secretion in different epithelia. Cases of complete NKCC1 deficiency in young patients are straightforward, leading to clinical presentations that overlap with the phenotypes observed in NKCC1 knockout mouse models. However, cases involving deleterious variants in one allele are more difficult, as the clinical presentation is variable, and the cause-effect relationship is not always clear. For instance, we worked on a single patient's case from multiple angles and published six related papers to convince ourselves of the cause-and-effect relationship between her NKCC1 mutation and her clinical presentations. The cluster of mutations in a small portion of the carboxyl terminus and its association with deafness point to a cause-and-effect relationship, even if the molecular mechanism is unknown. Overall, the preponderance of evidence suggests that the SLC12A2 gene is a human disease-causing and likely haploinsufficient gene that requires further investigation.

A novel mouse model for an inducible gene modification in the renal thick ascending limb.

The thick ascending limb (TAL) is critical for renal control of fluid and ion homeostasis. The function of the TAL depends on the activity of the bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2), which is highly abundant in the luminal membrane of TAL cells. TAL function is regulated by various hormonal and nonhormonal factors. However, many of the underlying signal transduction pathways remain elusive. Here, we describe and characterize a novel gene-modified mouse model for an inducible and specific Cre/Lox-mediated gene modification in the TAL. In these mice, tamoxifen-dependent Cre (CreERT2) was inserted into the 3'-untranslated region of the Slc12a1 gene, which encodes NKCC2 (Slc12a1-CreERT2). Although this gene modification strategy slightly reduced endogenous NKCC2 expression at the mRNA and protein levels, the lowered NKCC2 abundance was not associated with altered urinary fluid and ion excretion, urinary concentration, and the renal response to loop diuretics. Immunohistochemistry on kidneys from Slc12a1-CreERT2 mice revealed strong Cre expression exclusively in TAL cells but not in any other nephron portion. Cross-breeding of these mice with the mT/mG reporter mouse line showed a very low recombination rate (∼0% in male mice and <3% in female mice) at baseline but complete (∼100%) recombination after repeated tamoxifen administration in male and female mice. The achieved recombination encompassed the entire TAL and also included the macula densa. Thus, the new Slc12a1-CreERT2 mouse line allows inducible and very efficient gene targeting in the TAL and hence promises to be a powerful tool to advance our understanding of the regulation of TAL function.NEW & NOTEWORTHY The renal thick ascending limb (TAL) is critical for renal control of fluid and ion homeostasis. However, the underlying molecular mechanisms that regulate TAL function are incompletely understood. This study describes a novel transgenic mouse model (Slc12a1-creERT2) for inducible and highly efficient gene targeting in the TAL that promises to ease physiological studies on the functional role of candidate regulatory genes.

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

Molecular mechanisms, physiological roles, and therapeutic implications of ion fluxes in bone cells: Emphasis on the cation-Cl- cotransporters.

Bone turnover diseases are exceptionally prevalent in human and come with a high burden on physical health. While these diseases are associated with a variety of risk factors and causes, they are all characterized by common denominators, that is, abnormalities in the function or number of osteoblasts, osteoclasts, and/or osteocytes. As such, much effort has been deployed in the recent years to understand the signaling mechanisms of bone cell proliferation and differentiation with the objectives of exploiting the intermediates involved as therapeutic preys. Ion transport systems at the external and in the intracellular membranes of osteoblasts and osteoclasts also play an important role in bone turnover by coordinating the movement of Ca2+ , PO4 2- , and H+ ions in and out of the osseous matrix. Even if they sustain the terminal steps of osteoformation and osteoresorption, they have been the object of very little attention in the last several years. Members of the cation-Cl- cotransporter (CCC) family are among the systems at work as they are expressed in bone cells, are known to affect the activity of Ca2+ -, PO4 2- -, and H+ -dependent transport systems and have been linked to bone mass density variation in human. In this review, the roles played by the CCCs in bone remodeling will be discussed in light of recent developments and their potential relevance in the treatment of skeletal disorders.

High glucose-induced effects on Na+-K+-2Cl- cotransport and Na+/H+ exchange of blood-brain barrier endothelial cells: involvement of SGK1, PKCßII, and SPAK/OSR1.

Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCßII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCßII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCßII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCßII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCßII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

Multiple Facets and Roles of Na+-K+-Cl- Cotransport: Mechanisms and Therapeutic Implications.

The Na+-K+-Cl- cotransporters play key physiological and pathophysiological roles by regulating the membrane potential of many cell types and the movement of fluid across a variety of epithelial or endothelial structures. As such, they should soon become invaluable targets for the treatment of various disorders including pain, epilepsy, brain edema, and hypertension. This review highlights the nature of these roles, the mechanisms at play, and the unresolved issues in the field.

The role of HCO3- in propionate-induced anion secretion across rat caecal epithelium.

Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl- secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-induced Isc (IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl-- or nominally HCO3--free buffer. In the case of Cl-, the dependency was restricted to the presence of Cl- in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl--cotransporter, only numerically reduced IProp suggesting that a large part of this current must be carried by an ion other than Cl-. In the aboral caecum, IProp was significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IProp significantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3--cotransporters in caecum, which might underlie the observed Na+ dependency. These results suggest that propionate sensing in caecum is coupled to HCO3- secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen.

CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) Mediates Plant Resistance against Pseudomonas syringae.

Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na+:K+:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.

Targeting the WNK-SPAK/OSR1 Pathway and Cation-Chloride Cotransporters for the Therapy of Stroke.

Stroke is one of the major culprits responsible for morbidity and mortality worldwide, and the currently available pharmacological strategies to combat this global disease are scanty. Cation-chloride cotransporters (CCCs) are expressed in several tissues (including neurons) and extensively contribute to the maintenance of numerous physiological functions including chloride homeostasis. Previous studies have implicated two CCCs, the Na+-K+-Cl- and K+-Cl- cotransporters (NKCCs and KCCs) in stroke episodes along with their upstream regulators, the with-no-lysine kinase (WNKs) family and STE20/SPS1-related proline/alanine rich kinase (SPAK) or oxidative stress response kinase (OSR1) via a signaling pathway. As the WNK-SPAK/OSR1 pathway reciprocally regulates NKCC and KCC, a growing body of evidence implicates over-activation and altered expression of NKCC1 in stroke pathology whilst stimulation of KCC3 during and even after a stroke event is neuroprotective. Both inhibition of NKCC1 and activation of KCC3 exert neuroprotection through reduction in intracellular chloride levels and thus could be a novel therapeutic strategy. Hence, this review summarizes the current understanding of functional regulations of the CCCs implicated in stroke with particular focus on NKCC1, KCC3, and WNK-SPAK/OSR1 signaling and discusses the current and potential pharmacological treatments for stroke.

ROLE OF Β-ADRENOCEPTORS IN REGULATION OF ERYTHROCYTES' RHEOLOGICAL FUNCTIONS (REVIEW).

In this review, we discuss the role of adrenoceptors in the regulation of the rheological functions of erythrocytes. ß-adrenoceptors play an important role in the regulation of erythrocytes functions and metabolism. They participate in the modification of transport membrane proteins (Na+/K+-ATPase, Ca+2-ATPase, Na+/K+/2Cl-cotransporter, Na+/H+-antiporter, CAT-1, Ca2+-dependent K+ channels (Gard channels), the activity of adenylate cyclase and cAMP, AMP-dependent activation of the L-arginine/NOS system and erythrocyte NOS) and by this way modulate the cells volume, rheological properties (deformability, aggregability), intensity of NO synthesis and ATP reliase. These properties of erythrocytes determine, that, in addition to the transport of gases, they play the oxygen sensors role and can participate in the mechanisms of vasorelaxation and maintenance of a normal level of microcirculation.

Coordinate adaptations of skeletal muscle and kidney to maintain extracellular [K+] during K+-deficient diet.

Extracellular fluid (ECF) potassium concentration ([K+]) is maintained by adaptations of kidney and skeletal muscle, responses heretofore studied separately. We aimed to determine how these organ systems work in concert to preserve ECF [K+] in male C57BL/6J mice fed a K+-deficient diet (0K) versus 1% K+ diet (1K) for 10 days (n = 5-6/group). During 0K feeding, plasma [K+] fell from 4.5 to 2 mM; hindlimb muscle (gastrocnemius and soleus) lost 28 mM K+ (from 115 ± 2 to 87 ± 2 mM) and gained 27 mM Na+ (from 27 ± 0.4 to 54 ± 2 mM). Doubling of muscle tissue [Na+] was not associated with inflammation, cytokine production or hypertension as reported by others. Muscle transporter adaptations in 0K- versus 1K-fed mice, assessed by immunoblot, included decreased sodium pump α2-ß2 subunits, decreased K+-Cl- cotransporter isoform 3, and increased phosphorylated (p) Na+,K+,2Cl- cotransporter isoform 1 (NKCC1p), Ste20/SPS-1-related proline-alanine rich kinase (SPAKp), and oxidative stress-responsive kinase 1 (OSR1p) consistent with intracellular fluid (ICF) K+ loss and Na+ gain. Renal transporters' adaptations, effecting a 98% reduction in K+ excretion, included two- to threefold increased phosphorylated Na+-Cl- cotransporter (NCCp), SPAKp, and OSR1p abundance, limiting Na+ delivery to epithelial Na+ channels where Na+ reabsorption drives K+ secretion; and renal K sensor Kir 4.1 abundance fell 25%. Mass balance estimations indicate that over 10 days of 0K feeding, mice lose ~48 µmol K+ into the urine and muscle shifts ~47 µmol K+ from ICF to ECF, illustrating the importance of the concerted responses during K+ deficiency.

Sex differences in solute transport along the nephrons: effects of Na+ transport inhibition.

Each day, ~1.7 kg of NaCl and 180 liters of water are reabsorbed by nephron segments in humans, with urinary excretion fine tuned to meet homeostatic requirements. These tasks are coordinated by a spectrum of renal Na+ transporters and channels. The goal of the present study was to investigate the extent to which inhibitors of transepithelial Na+ transport (TNa) along the nephron alter urinary solute excretion and how those effects may vary between male and female subjects. To accomplish that goal, we developed sex-specific multinephron models that represent detailed transcellular and paracellular transport processes along the nephrons of male and female rat kidneys. We simulated inhibition of Na+/H+ exchanger 3 (NHE3), bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2), Na+-Cl- cotransporter (NCC), and amiloride-sensitive epithelial Na+ channel (ENaC). NHE3 inhibition simulations predicted a substantially reduced proximal tubule TNa, and NKCC2 inhibition substantially reduced thick ascending limb TNa. Both gave rise to diuresis, natriuresis, and kaliuresis, with those effects stronger in female rats. While NCC inhibition was predicted to have only minor impact on renal TNa, it nonetheless had a notable effect of enhancing excretion of Na+, K+, and Cl-, particularly in female rats. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na+ (increased) and K+ (decreased) and to have only a minor impact on whole kidney TNa. Unlike inhibition of other transporters, ENaC inhibition induced stronger natriuresis and diuresis in male rats than female rats. Overall, model predictions agreed well with measured changes in Na+ and K+ excretion in response to diuretics and Na+ transporter mutations.