Extracellular Tonicity Research Articles

WNK1 is a central osmolality sensor for arginine vasopressin release and acts through the OSR1/SPAK kinase cascade

Background: Maintaining internal osmolality constancy and water homeostasis is essential for life. The circumventricular organs (CVOs) of the brain, including the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO), lack a blood-brain barrier. Neurons in OVLT and SFO detect increases in serum osmolality that stimulate the production of vasopressin (AVP) in paraventricular nuclei (PVN) to be released in the posterior pituitary. We have recently reported that WNK1 in sensory neurons in CVOs functions as an osmolality sensor for AVP release (Jin et al., JCI, 2023). WNK1 activates Kv3.1 to increase action potential, traveling down to PVN to increase AVP synthesis for release from the posterior pituitary. Here, we propose that OSR1/SPAK acts as the downstream kinase for WNK1 in regulating AVP release. We further investigated the regulation of vasopressin release by the WNK1-OSR1/SPAK cascade in response to extracellular hyperosmolality. Methods: Cre recombinase-carrying retrograde AAV virus was stereotaxically injected into PVN nuclei from normal mice and SPAK-null mice with a homozygous OSR1-floxed allele (SAPK−/−;OSR1flox/flox). Metabolic cage studies were performed on mice. Urine output, water intake, serum osmolality, serum AVP, and copeptin levels were measured. K+ currents were measured from the OVLT neurons by whole-cell patch clamp. Results: Increased osmolality, either by water restriction or mannitol injection, activated OSR1/SPAK in OVLT, as evident by increased serine-373 phospho-SPAK and serine-325 phospho-OSR. Like the WNK1 deletion, the double deletion of OSR/SPAK in OVLT caused polyuria with decreased urine osmolality that persisted in water restriction. Circulating levels of AVP and copeptin were increased by water restriction in control mice. In contrast, water restriction failed to increase the levels in OSR/SPAK-deletion mice. Knockdown of the Kv3.1 channel in OVLT by shRNA showed the central diabetes insipidus (DI) phenotype as well. Hypertonicity increased K+ currents and WNKs inhibitor prevented the increase in OVLT neurons. Conclusions: Mice with neuronal-specific OSR1/SPAK deletion have water homeostasis defects consistent with partial central DI; Evidence supporting that OSR1/SPAK acts downstream of WNK1 to regulate AVP release; WNK1-OSR1/SPAK activates Kv3.1 to increase firing frequency of neurons projecting from CVOs to PVN. Our findings reveal that an intracellular protein acts as a sensor for extracellular tonicity and provide fresh insights into the mechanisms by which the body maintains osmolality constancy. Future studies will investigate how OSR1/SPAK regulates Kv3.1 channels. NIDDK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2.

ABSTRACTNFAT5 is the only known mammalian tonicity-responsive transcription factor with an essential role in cellular adaptation to hypertonic stress. It is also implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity, but the underlying mechanisms remain elusive. Here, we demonstrate that NFAT5 enters the nucleus via the nuclear pore complex. We found that NFAT5 utilizes a unique nuclear localization signal (NFAT5-NLS) for nuclear import. siRNA screening revealed that only karyopherin β1 (KPNB1), but not karyopherin α, is responsible for the nuclear import of NFAT5 via direct interaction with the NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is driven by exportin-T (XPOT), where the process requires RuvB-like AAA-type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified an unconventional tonicity-dependent nucleocytoplasmic trafficking pathway for NFAT5 that represents a critical step in orchestrating rapid cellular adaptation to change in extracellular tonicity. These findings offer an opportunity for the development of novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

MiR-324-5p and miR-30c-2-3p Alter Renal Mineralocorticoid Receptor Signaling under Hypertonicity.

The Mineralocorticoid Receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron, but mechanisms regulating MR expression are still poorly understood. We previously showed that RNA Binding Proteins (RBPs) regulate MR expression at the post-transcriptional level in response to variations of extracellular tonicity. Herein, we highlight a novel regulatory mechanism involving the recruitment of microRNAs (miRNAs) under hypertonicity. RT-qPCR validated miRNAs candidates identified by high throughput screening approaches and transfection of a luciferase reporter construct together with miRNAs Mimics or Inhibitors demonstrated their functional interaction with target transcripts. Overexpression strategies using Mimics or lentivirus revealed the impact on MR expression and signaling in renal KC3AC1 cells. miR-324-5p and miR-30c-2-3p expression are increased under hypertonicity in KC3AC1 cells. These miRNAs directly affect Nr3c2 (MR) transcript stability, act with Tis11b to destabilize MR transcript but also repress Elavl1 (HuR) transcript, which enhances MR expression and signaling. Overexpression of miR-324-5p and miR-30c-2-3p alter MR expression and signaling in KC3AC1 cells with blunted responses in terms of aldosterone-regulated genes expression. We also confirm that their expression is increased by hypertonicity in vivo in the kidneys of mice treated with furosemide. These findings may have major implications for the pathogenesis of renal dysfunctions, sodium retention, and mineralocorticoid resistance.

Study of transmembrane ion transport under tonicity imbalance using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model

Transmembrane ion transport under tonicity imbalance has been investigated using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model, by considering the cell diameter d [m] and the initial intracellular ion concentration c in [mM] as a function of tonicity expressed by sucrose concentration c s [mM]. The transmembrane ion transport is influenced by extracellular tonicity conditions, leading to a facilitation/inhibition of ion passage through the cell membrane. The transmembrane transport coefficient P [m s−1], which represents the ability of transmembrane ion transport, is calculated by the extracellular ion concentrations obtained by improved ion transport model and LF-EIS measurement. P is calculated as 4.11 × 10−6 and 3.44 × 10−6 m s−1 at c s of 10 and 30 mM representing hypotonic condition, 2.44 × 10−6 m s−1 at c s of 50 mM representing isotonic condition, and 3.68 × 10−6, 5.16 × 10−6 , 9.51 × 10−6, and 14.89 × 10−6 m s−1 at c s of 75, 100, 125 and 150 mM representing hypertonic condition. The LF-EIS results indicate that the transmembrane ion transport is promoted under hypertonic and hypotonic conditions compared to isotonic condition. To verify the LF-EIS results, fluorescence intensity F [–] of extracellular potassium ions is observed to obtain the temporal distribution of average potassium ion concentration within the region of 3.6 μm from cell membrane interface c ROI [mM]. The slopes of ∆c ROI /c ROI1 to time t are 0.0003, 0.0002, and 0.0006 under hypotonic, isotonic, and hypertonic conditions, where c ROI1 denotes initial c ROI , which shows the same tendency with LF-EIS result that is verified by the potassium ion fluorescence observation.

A study on alterations in membrane physiology of human erythrocytes due to extracellular tonicity through dielectrophoresis

To get an insight into the physiological reality of biological cells it is necessary to obtain knowledge regarding their electrical make. It is in this view an attempt has been made to study the dielectric property of human erythrocytes suspended in solutions of different tonicity (Isotonic, Hypertonic, and Hypotonic) using the technique of Dielectrophoresis. This technique is concerned with the electrical constitution of the cell and is sensitive to small perturbations in cell physiology. In the present study, Erythrocytes suspended in different prepared solutions are subjected to Non- Uniform Electric Field produced by Pin-Pin Electrode configuration.

WNK3 and WNK4 exhibit opposite sensitivity with respect to cell volume and intracellular chloride concentration.

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.

Red blood cells under varying extracellular tonicity conditions: an optical tweezers combined with micro-Raman study

Extracellular tonicity has a significant influence on human red blood cell deformation capability. Advancements in the area of laser physics and optical trapping have opened up a plethora of applications for understanding cell structure and dynamics. Here, Raman Tweezers technique was employed to investigate the impact of extracellular tonicity by exposing human red blood cells to both hypertonic and hypotonic intravenous fluids. Heme aggregation was observed in hypertonic saline solution, accompanied with damage in membrane protein. Loss of intracellular hemoglobin in hypotonic solution was evident from the decrease in porphyrin breathing mode present at 752 cm−1. Oxygen binding to the central iron in the red blood cell heme was also affected under both hyper/hypo tonicity conditions. Morphological deviation of discocytes to echinocytes/spherocytes were also evident from quantitative phase imaging. Principal component analysis have showed clear differentiation of samples in order to classify the control erythrocytes and the tonicity stressed erythrocytes. Present study has also demonstrated the application of Raman Tweezers spectroscopy as a potential tool for probing red blood cell under different stress conditions.

Silver ions promote blebs growth in U251 glioblastoma cell by activating nonselective cationic currents

Glioblastoma (GBM) is the most common and aggressive human brain cancer with low prognosis and therefore the discovery of new anticancer agents is needful. Sulfydryl reagents, such as silver, have been shown to induce membrane vesiculation in several cellular models through a mechanism that has not been yet completely clarified. Using U251 glioblastoma cells, we observed that silver induced irreversible bleb formation of the plasma membrane. This morphological event was anticipated by an increase of intracellular Ca2+ associated to extracellular Ca2+ influx. Accordingly, using patch-clamp whole cell recording during silver ion application, inward current/s (IAg) at −90 mV were detected and cells were permeable to Ca2+ and monovalent ions such as Na+. IAg activation and the intracellular Ca2+ increase promoted by silver ions (Ag+) were prevented by co-application of 20 µM cysteine and 300 µM DIDS (4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid), suggesting a critical role of thiol groups in the biological effects of silver ions. IAg was partially inhibited by 1 mM Gd3+, an unspecific inhibitor of cationic currents. Cysteine, Gd3+ and extracellular free Ca2+ solution completely abolished blebbing formation promoted by Ag+. Furthermore, extracellular Na+ ion replacement with TEA or an increase of extracellular tonicity by sucrose (100 mM) reduced both size and growth of membrane blebbing. Our data suggest that Ag+ promotes the formation necrotic blebs as consequence of the increase of intracellular Ca2+ and intracellular hydrostatic pressure associated to the activation of cationic currents. Since silver-induced blebs were less evident in benign glial human Müller MIO-M1 cells, silver compounds could represent new adjuvant to anticancer agents to improve GBM therapies.

Heat acclimatization blunts copeptin responses to hypertonicity from dehydrating exercise in humans.

Acclimatization favors greater extracellular tonicity from lower sweat sodium, yet hyperosmolality may impair thermoregulation during heat stress. Enhanced secretion or action of vasopressin could mitigate this through increased free water retention. Aims were to determine responses of the vasopressin surrogate copeptin to dehydrating exercise and investigate its relationships with tonicity during short and long‐term acclimatization. Twenty‐three participants completed a structured exercise programme following arrival from a temperate to a hot climate. A Heat Tolerance Test (HTT) was conducted on Day‐2, 6, 9 and 23, consisting of 60‐min block‐stepping at 50% VO 2peak, with no fluid intake. Resting sweat [Na+] was measured by iontophoresis. Changes in body mass (sweat loss), core temperature, heart rate, osmolality (serum and urine) and copeptin and aldosterone (plasma) were measured with each Test. From Day 2 to Day 23, sweat [Na+] decreased significantly (adjusted P < 0.05) and core temperature and heart rate fell. Over the same interval, HTT‐associated excursions were increased for serum osmolality (5 [−1, 9] vs. 9 [5, 12] mosm·kg−1), did not differ for copeptin (9.6 [6.0, 15.0] vs. 7.9 [4.3, 14.7] pmol·L−1) and were reduced for aldosterone (602 [415, 946] vs. 347 [263, 537] pmol·L−1). Urine osmolality was unchanging and related consistently to copeptin at end‐exercise, whereas the association between copeptin and serum osmolality was right‐shifted (P = 0.0109) with acclimatization. Unchanging urine:serum osmolality argued against increased renal action of vasopressin. In conclusion, where exercise in the heat is performed without fluid replacement, heat acclimatization does not appear to enhance AVP‐mediated free water retention in humans.

WNK3 and WNK4 Exhibit Opposite Sensibility for Cell Volume and Intracellular Chloride Concentration

BackgroundThe intracellular chloride concentration [Cl−]i and cell volume modulates the activity of the electroneutral cation‐coupled chloride cotransporters (CCCs) such as the NKCCs and the KCCs. Cell shrinkage or reduction of [Cl−]i induce phosphorylation of CCCs, which activates NKCCs and inhibits KCCs, promoting ion influx, while cell swelling or increase of [Cl−]i induces dephosphorylation, that inhibits NKCCs and activates KCCs, stimulating ion efflux. We have shown that the effect of [Cl−]i towards CCCs is traduced by the WNK1 or WNK4 kinases. However, cell volume and [Cl−]i moves in opposite directions. Cell shrinkage increases [Cl−]i and cell swelling decreases [Cl−]i. Thus, the effect of cell volume towards CCCs must be traduced by a different kinase. Because WNK3 bypasses the tonicity requirements for regulation of the CCCs (de Los Heros, PNAS 2006), we tested the hypothesis that WNK3 is sensitive to cell volume, rather than to the [Cl−]i.MethodsXenopus oocytes were microinjected with the Na‐Cl cotransporter (NCC) or the K‐Cl cotransporter (KCC4) cRNA alone or together with WNK3 or WNK4 wild type, or mutants in which catalytic activity and/or the chloride‐binding site has been eliminated (WNK3‐DA; WNK3‐L295/297F; WNK3‐DA‐LLFF; WNK4‐DA; WNK4‐L322F) or with chimeric constructs obtained by swapping the amino, kinase or carboxyl terminal domains between WNK3 and WNK4. The effect of [Cl−]i or extracellular tonicity on WNKs effects on NCC or KCC4 was assessed by tracer 22Na+ or 86Rb+ uptake, respectively. The effect on extracellular tonicity on WNKs activity was assessed as WNK phosphorylation at the activating serine of the T‐loop with specific antibodies by Western blot, as a surrogate of activity.ResultsDepletion of [Cl−]i increases the activity of WNK4 and thus increases the NCC activity. Elimination of WNK4 chloride‐binding site turned it into a constitutively active kinase toward NCC. In contrast the degree of activation of NCC by WNK3 was not affected by reduction of [Cl−]i and elimination of the chloride‐binding site in WNK3 had no effect on the activity of the kinase. Compared to isotonic condition, WNK3 phosphorylation significantly diminished or increased by 50% in hypotonic or hypertonic conditions, respectively. The phosphorylation of WNK4 was not affected by similar changes in tonicity. Chimeric proteins between WNK3 and WNK4 revealed that the carboxyl terminal domain modulates chloride affinity in WNKs. That is, swapping the WNK4 carboxyl terminal domain into WNK3 amino and kinase domain rendered this construct sensitive to changes in the [Cl−]i.DiscussionWe previously demonstrated that WNK4 is sensitive to [Cl−]i (Bazúa‐Valenti, JASN 2015). Here we show that WNK3 is not sensitive to changes of the [Cl−]i, but is modulated by changes in cell volume. In addition, WNK3, but not WNK4, phosphorylation was sensitive to changes in cell volume. Hypotonicity reduces WNK3 phosphorylation, while hypertonicity increased it. Thus, we propose that while WNK4 is a chloride sensitive kinase, WNK3 is a volume sensitive kinase, modulating the effect of cell volume changes towards CCCs activity.Support or Funding InformationFronteras de la Ciencia Grant No. 23 from Conacyt to GGThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NFAT5 participates in seawater inhalation-induced acute lung injury via modulation of NF-κB activity

Nuclear factor of activated T cells 5 (NFAT5) is a transcription factor that can be activated by extracellular tonicity. It has been reported that NFAT5 may increase the transcription of certain osmoprotective genes in the renal system, and the aim of the current study was to explore the role of NFAT5 in seawater inhalation-induced acute lung injury. Though establishing the model of seawater inhalation-induced acute lung injury, it was demonstrated that seawater inhalation enhanced the transcription and protein expression of NFAT5 (evaluated by reverse transcription-polymerase chain reaction, immunohistochemistry stain and western blotting) and activation of nuclear factor (NF)-κB (evaluated by western blotting and mRNA expression levels of three NF-κB-dependent genes) both in lung tissue and rat alveolar macrophage cells (NR8383 cells). When expression of NFAT5 was reduced in NR8383 cells using an siRNA targeted to NFAT5, the phosphorylation of NF-κB and transcription of NF-κB-dependent genes were significantly reduced. In addition, the elevated content of certain inflammatory cytokines [tumor necrosis factor α, interleukin (IL)-1 and IL-8] were markedly reduced. In conclusion, NFAT5 serves an important pathophysiological role in seawater inhalation-induced acute lung injury by modulating NF-κB activity, and these data suggest that NFAT5 may be a promising therapeutic target.

Structural Determinants of Oligomerization of the Aquaporin-4 Channel

The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.

Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways

Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.

PP.11.18

Objective: Evidences indicate that in addition to long term regulation of blood pressure, in the kidney resides the initial trigger for the development of hypertension. Altered capacity of the kidney to excrete sodium and water in relation to intake has been proposed as a basic mechanism of initiating hypertension. Betaine is one of the major organic osmolytes and the upregulation of its betaine/gamma-aminobutyric acid transporter (BGT1) in the renal medulla is related to high extracellular tonicity and urinary osmolality. Design and method: The present study investigated the abnormalities in water and sodium balance and osmoregulation in a model of polygenic multifactorial arterial hypertension, the spontaneously hypertensive rats (SHR, n = 58) and compared with their normotensive controls Wistar Kyoto (WKY, n = 46 from their prehypertensive phase (4 weeks of age) until the development hypertension and of organ damage (28–30 weeks of age). Rats were housed in metabolic cages to monitor water and salt balance parameters at the age of 4–5, 14–16 and 28–30 weeks of age. Results: No difference of blood pressure (both indirectly tail cuff method and invasively, as well heart rate, body weight, serum sodium, potassium and creatinine levels) was measured at the age of 5 weeks of age between SHR and WKY. Daily urinary volume was reduced in prehypertensive SHR as compared to WKY (-36 %, p Conclusions: All these results suggest in prehypertensive SHR an early positive balance of water before sodium balance alteration.

Central Pontine Myelinolysis: A Case Study

Abstract Central Pontine Myelinolysis (CPM) commonly presents as a complication of treatment in patients with profound life threatening hyponatraemia. It occurs when the sodium level is corrected too rapidly. Hyponatraemia should never be corrected at a rate greater than 8-10mmol/L of sodium per day. Rapid correction causes extracellular tonicity and will continue to drive water out of the brain’s cells leading to cellular dysfunction. Frequent clinical signs include dysphagia, dysarthria, diplopia and acute para/quadraparesis. Patients can also experience locked in syndrome, where cognitive function is intact but all muscles are paralysed with the exception of eye blinking. CPM gets its name as it occurs when cell dysfunction causes destruction of the myelin sheath of nerve cells in the brain stem, more specifically the pons. It is associated with poor prognosis and prevention is of primary importance. Freddy is a 35 year old freelance graphic designer, fitness instructor and ultra-marathon runner. In October 2011 he competed in the Sahara Marathon in Morocco, a 6 day 255km ultra-marathon. At the end of the third day Freddy was found collapsed and vomiting. He was confused and was suffering severe leg cramps. The next morning Freddy suffered a single convulsive episode and was subsequently transferred to a hospital in Egypt.

Angiogenin-cleaved tRNA halves interact with cytochrome c, protecting cells from apoptosis during osmotic stress.

Adaptation to changes in extracellular tonicity is essential for cell survival. However, severe or chronic hyperosmotic stress induces apoptosis, which involves cytochrome c (Cyt c) release from mitochondria and subsequent apoptosome formation. Here, we show that angiogenin-induced accumulation of tRNA halves (or tiRNAs) is accompanied by increased survival in hyperosmotically stressed mouse embryonic fibroblasts. Treatment of cells with angiogenin inhibits stress-induced formation of the apoptosome and increases the interaction of small RNAs with released Cyt c in a ribonucleoprotein (Cyt c-RNP) complex. Next-generation sequencing of RNA isolated from the Cyt c-RNP complex reveals that 20 tiRNAs are highly enriched in the Cyt c-RNP complex. Preferred components of this complex are 5' and 3' tiRNAs of specific isodecoders within a family of isoacceptors. We also demonstrate that Cyt c binds tiRNAs in vitro, and the pool of Cyt c-interacting RNAs binds tighter than individual tiRNAs. Finally, we show that angiogenin treatment of primary cortical neurons exposed to hyperosmotic stress also decreases apoptosis. Our findings reveal a connection between angiogenin-generated tiRNAs and cell survival in response to hyperosmotic stress and suggest a novel cellular complex involving Cyt c and tiRNAs that inhibits apoptosome formation and activity.

The ideal crystalloid – what is ‘balanced’?

This review explores the contemporary definition of the term 'balanced crystalloid' and outlines optimal design features and their underlying rationale. Crystalloid interstitial expansion is unavoidable, but also occurs with colloids when there is endothelial glycocalyx dysfunction. Reduced chloride exposure may lessen kidney dysfunction and injury with a possible mortality benefit. Exact balance from an acid-base perspective is achieved with a crystalloid strong ion difference of 24 mEq/l. This can be done simply by replacing 24 mEq/l of chloride in 0.9% sodium chloride with bicarbonate or organic anion bicarbonate substitutes. Potassium, calcium and magnesium additives are probably unnecessary. Large volumes of mildly hypotonic crystalloids such as lactated Ringer's solution reduce extracellular tonicity in volunteers and increase intracranial pressure in nonbrain-injured experimental animals. A total cation concentration of 154 mmol/l with accompanying anions provides isotonicity. Of the commercial crystalloids, Ringer's acetate solution is close to balanced from both acid-base and tonicity perspectives, and there is little current evidence of acetate toxicity in the context of volume loading, in contrast to renal replacement. The case for balanced crystalloids is growing but unproven. A large randomized controlled trial of balanced crystalloids versus 0.9% sodium chloride is the next step.

A Clinical Approach to the Treatment of Chronic Hypernatremia

Hypernatremia is a commonly encountered electrolyte disorder occurring in both the inpatient and outpatient settings. Community-acquired hypernatremia typically occurs at the extremes of age, whereas hospital-acquired hypernatremia affects patients of all age groups. Serum sodium concentration is linked to water homeostasis, which is dependent on the thirst mechanism, arginine vasopressin, and kidney function. Because both hypernatremia and the rate of correction of hypernatremia are associated with significant morbidity and mortality, prompt effective treatment is crucial. Chronic hypernatremia can be classified into 3 broad categories, hypovolemic, euvolemic, and hypervolemic forms, with each form having unique treatment considerations. In this teaching case, we provide a clinically based quantitative approach to the treatment of both hypervolemic and hypovolemic hypernatremia, which occurred in the same patient during the course of a prolonged illness.

CDK9 regulates hypotonicity‐induced nuclear export of NFAT5

NFAT5, also known as Osmotic Response Element‐binding Protein (OREBP), or Tonicity‐responsive Enhancer‐binding Protein (TonEBP), is an osmo‐ sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. The subcellular localization and activity of NFAT5 are tightly regulated by extracellular tonicity. Earlier we showed that hypotoncity‐induced nuclear export of NFAT5 is regulated by the sequential phosphorylation of two serine residues (ser‐155 and ser‐158), and we also identified Casein Kinase 1 (CK1) as the ser‐158 kinase. In this study, using protein fractionations, in‐vitro kinase assay and mass spectrometric protein identification approaches, we identified Cyclin‐dependent Kinase 9 (CDK9) as a putative ser‐155 kinase. NFAT5 is co‐immunoprecipitated with CDK9 and its regulatory subunit cyclin T. Furthermore, CDK9 inhibitors and siRNAs significantly diminished NFAT5 nuclear export in response to hypotonicity.

Volume regulation in mammalian skeletal muscle: the role of sodium–potassium–chloride cotransporters during exposure to hypertonic solutions

Controversy exists as to whether mammalian skeletal muscle is capable of volume regulation in response to changes in extracellular osmolarity despite evidence that muscle fibres have the required ion transport mechanisms to transport solute and water in situ. We addressed this issue by studying the ability of skeletal muscle to regulate volume during periods of induced hyperosmotic stress using single, mouse extensor digitorum longus (EDL) muscle fibres and intact muscle (soleus and EDL). Fibres and intact muscles were loaded with the fluorophore, calcein, and the change in muscle fluorescence and width (single fibres only) used as a metric of volume change. We hypothesized that skeletal muscle exposed to increased extracellular osmolarity would elicit initial cellular shrinkage followed by a regulatory volume increase (RVI) with the RVI dependent on the sodium–potassium–chloride cotransporter (NKCC). We found that single fibres exposed to a 35% increase in extracellular osmolarity demonstrated a rapid, initial 27–32% decrease in cell volume followed by a RVI which took 10-20 min and returned cell volume to 90–110% of pre-stimulus values. Within intact muscle, exposure to increased extracellular osmolarity of varying degrees also induced a rapid, initial shrinkage followed by a gradual RVI, with a greater rate of initial cell shrinkage and a longer time for RVI to occur with increasing extracellular tonicities. Furthermore, RVI was significantly faster in slow-twitch soleus than fast-twitch EDL. Pre-treatment of muscle with bumetanide (NKCC inhibitor) or ouabain (Na+,K+-ATPase inhibitor), increased the initial volume loss and impaired the RVI response to increased extracellular osmolarity indicating that the NKCC is a primary contributor to volume regulation in skeletal muscle. It is concluded that mouse skeletal muscle initially loses volume then exhibits a RVI when exposed to increases in extracellular osmolarity. The rate of RVI is dependent on the degree of change in extracellular osmolarity, is muscle specific, and is dependent on the functioning of the NKCC and Na+, K+-ATPase.