Whole-body electrolyte-free water clearance: derivation and clinical utility in analyzing the pathogenesis of the dysnatremias

The total exchangeable sodium (Na_e), total exchangeable potassium (K_e), and total body water (TBW) are the major determinants of the plasma water sodium concentration ([Na⁺]_pw). The relationship between [Na⁺]_pw and Na_e, K_e, and TBW was empirically determined by Edelman et al., where: [Na⁺]_pw = 1.11(Na_e + K_e)/TBW − 25.6 (Eq. 1). According to Eq. 1, changes in the mass balance of Na⁺, K⁺, and H₂O will therefore result in changes in the [Na⁺]_pw. Historically, in evaluating the pathogenesis of the dysnatremias, free water clearance (FWC) and electrolyte-free water clearance (EFWC) have been used to evaluate the pathophysiology of the dysnatremias. However, such analyses are only valid when there is no concomitant input and non-renal output of Na⁺, K⁺, and H₂O. Since the classic FWC and EFWC formulas fail to account for the input and non-renal output of Na⁺, K⁺, and H₂O, these formulas cannot be used to evaluate the pathogenesis of the dysnatremias or to predict the directional change in the [Na⁺]_pw. In this article, we have addressed this limitation by deriving a new formula, termed whole-body electrolyte-free water clearance (WB-EFWC), which calculates whole-body electrolyte-free water clearance for a given mass balance of Na⁺, K⁺, and H₂O, rather than simply the urinary component (FWC, EFWC formulas). Unlike previous formulas, which consider only the renal component of electrolyte-free water clearance, WB-EFWC accounts for all sources of input and output of Na⁺, K⁺, and H₂O, and will therefore be helpful in conceptually understanding the basis for changes in the [Na⁺]_pw in patients with the dysnatremias.     

Role of potassium in hypokalemia-induced hyponatremia: lessons learned from the Edelman equation

It is well known that changes in the mass balance of K⁺ can lead to an alteration in the plasma water sodium concentration ([Na⁺ ]_pw). We have recently shown that based on the Edelman equation, the [Na⁺ ]_pw is determined by the total exchangeable Na⁺ (Na_e), total exchangeable K⁺ (K_e), total body water (TBW), osmotically inactive Na_e and K_e, plasma water [K⁺], intracellular and extracellular osmotically active non-Na⁺ and non-K⁺ osmoles, and plasma osmotically active non-Na⁺ and non-K⁺ osmoles. In light of these findings, a re-analysis of the role of K⁺ in modulating the [Na⁺]_pw is required in understanding the pathophysiology of hypokalemia-induced hyponatremia. In this article, we characterize the complex role of K⁺ in the pathogenesis of hypokalemia-induced hyponatremia using a three-compartment model and the known parameters in the Edelman equation. Our analysis indicates that K⁺ modulates the [Na⁺]_pw by changing K_e in addition to the parameters in the y-intercept of the Edelman equation. Moreover, the magnitude of potassium-induced changes in the [Na⁺]_pw is determined by the pathophysiologic mechanisms by which changes in K_e occur.     

A simple quantitative approach to analyzing the generation of the dysnatremias

Background. Although the dysnatremias are the most common electrolyte disorders in hospitalized patients, the complexity of the parameters normally used to explain their generation mechanistically is often bewildering to medical students and experts alike. A number of methods have been utilized clinically to analyze retrospectively and predict prospectively the pathogenesis of these disorders. These approaches include the measurements of plasma and urine osmolality, free water clearance, electrolyte free water clearance, and tonicity balance.Methods. All previous analyses are problematic in that they fail to incorporate mathematically in a single equation the known factors that account quantitatively for changes in the plasma water sodium concentration. In this paper, we have derived a simple formula for use at the bedside based on all known factors that can generate the dysnatremias. The formula incorporates (1) the known empirical relationship between the plasma water Na⁺ concentration, total body water (TBW), and exchangeable Na⁺ (Na⁺_e) and K⁺ (K⁺_e); (2) changes in mass balance of H₂O (V_MB) and Na⁺ + K⁺ (E_MB); and (3) the effect of hyperglycemia.Results. This new equation, unlike all previous qualitative and quantitative approaches, can account mathematically for the simultaneous effects of TBW, Na_e, K_e, E_MB, V_MB, and the plasma glucose on the plasma water sodium concentration. Clinical examples are provided that demonstrate the utility of this new equation in analyzing the pathogenesis of the dysnatremias.Conclusion. The conceptual simplification resulting from the use of this formula should significantly improve the current approaches used in analyzing the generation of the dysnatremias.     

α 1‐Adrenoceptor‐mediated Negative Inotropic Effect Caused by Intracellular Ionic Activities in Guinea‐pig Papillary Muscle

In guinea‐pig papillary muscle, phenylephrine (PE), an agonist of α₁‐adrenoceptor (α₁‐AR), led to a transient negative inotropic effect (NIE) and a subsequent sustained positive inotropic effect (PIE). To clarify the ionic mechanisms underlying the NIE, we measured the [Na⁺]i or [pH]i by ion‐selective microelectrodes. PE produced a decrease in the intracellular Na⁺ concentration ([Na⁺]i) and an increase in intracellular pH ([pH]i). During the phase of NIE, PE produced only a (−) change of [Na⁺]i (Δ[Na⁺]i). With a decrease in extracellular Na⁺ or an increase in extracellular Ca²⁺, the PE‐induced NIE was attenuated and PE produced (+)Δ[Na⁺]i. The PE‐induced NIE and (−)Δ[Na⁺]i were definitely strengthened by lowering the bath temperature or increasing the stimulation frequency. 2‐(2,6‐di‐methoxyphenoxyethyl)amino‐methyl‐1,4‐benzidioxane HCl, an antagonist of α_1A‐AR, completely abolished the PE‐induced NIE and (−)Δ[Na⁺]i. Phorbol 12,13‐dibutyrate, an activator of protein kinase C (PKC), decreased the baseline [Na⁺]i and twitch force and increased the baseline [pH]i in mimicry of PE. Pretreatment with 1‐5(isoquinolinesulphonyl)‐2‐methylpiperazine, an inhibitor of PKC, abolished the PE‐induced NIE and (−)Δ[Na⁺]i. During pretreatment with benzamil, an inhibitor of Na⁺/Ca²⁺ exchange, we found that the PE‐induced NIE and (−)Δ[Na⁺]i were reversibly abolished. Our results indicate the PE‐induced NIE may be elicited upon the activation of Na⁺/Ca²⁺ exchange which can be attributed to the (−)Δ[Na⁺]i. (−)Δ[Na⁺]i is mediated through a PKC‐dependent pathway via an activation of α_1A‐AR subtype and its effect could be strengthened remarkably at high [Na⁺]i and [Ca²⁺]i values.     

Serum Sodium: A Reliable and Validated Predictor for Mortality in Enteric Fistula Patients Complicated with Sepsis

Purpose: The aim was to evaluate the predictive value of serial serum sodium determination for mortality in enteric fistula (EF) patients complicated with sepsis. Methods: Between January 1^st, 2012 to January 13^th, 2013, we performed a prospective observational study enrolling 162 patients. Patients were divided into survivors group (n = 119) and nonsurvivors group (n = 43) according to 28-day outcomes. Laboratory variables on day 0, day 3, and day 7 after admission were recorded. [Na⁺]₀ was defined as serum [Na⁺] value on admission. [Na⁺]₃ was defined as serum [Na⁺] value on day 3. Δ [Na⁺]₃ was defined as changes from [Na⁺]₃ to [Na⁺]₀. The definition applied to other parameters_. The results were validated in an independent cohort of 116 patients. Results: ROC analysis showed that [Na⁺]₇>147.5 mmol/L and ΔNa₇>5.2 mmol/L were reliable predictors ([Na⁺]₇: 81.2% sensitivity, 87.7% specificity, (area under the curve(AUC):0.872, p < .001; Δ[Na⁺]₇: 81.3% sensitivity, 83.6% specificity, AUC:0.836, p < .001) for mortality. The combination form ([Na⁺]₇>147.5 mmol/L+ Δ[Na]₇>5.2 mmol/L+ ΔPCT₇<5.3 ng/ml) had greatest predictive value (AUC:0.899, p < .001). Their predictive values were confirmed in the validation cohort. Conclusions: Serum sodium was a reliable predictor for mortality in abdominal septic patients, which should be paid close attention in the critical care.     

Renal tubular acidosis

The term renal tubular acidosis (RTA) is applied to a group of transport defects in the reabsorption of bicarbonate (HCO ₃ ^– ), the excretion of hydrogen ions, or both. On clinical and pathophysiological grounds, RTA can be separated into three main types: distal RTA (type 1), proximal RTA (type 2) and hyperkalaemic RTA (type 4). Some patients present combined types of proximal and distal RTA or of hyperkalaemic and distal RTA. Diagnosis of RTA should be suspected when a patient presents a normal plasma anion gap, and hyperchloraemic metabolic acidosis. A normal plasma anion gap (Na⁺–[Cl^–+HCO₃ ^–]=8–16 mEq/l) reflects loss of HCO₃ ^– from the extracellular fluid via the gastro-intestinal tract or the kidney, dilution of extracellular buffer or administration of hydrochloric acid (HCl) or its precursors. Distinction of RTA from other disorders is greatly facilitated by the study of the urine anion gap (Na⁺+K⁺–Cl^–). This index estimates the urinary concentration of ammonium in a patient with hyperchloraemic metabolic acidosis. A negative urine anion gap (Cl^–Na⁺+K⁺) suggests the presence of gastro-intestinal or renal loss of HCO₃ ^–, while a positive urine anion gap (Cl^–++K⁺) is indicative of a distal acidification defect. Determination of plasma potassium, of urine pH at low plasma HCO₃ ^– concentration, and of urineP co ₂ and fractional excretion of HCO₃ ^– at normal plasma HCO₃ ^– concentration permits the differentiation between the various types of RTA.     

The effect of antenatal dexamethasone administration on glomerular filtration rate and renal sodium excretion in premature infants

Creatinine clearance (C_cr) and renal sodium (Na⁺) excretion were measured in 10 premature infants (gestational age <34 weeks) whose mothers had received dexamethasone before delivery (group D) and in 11 whose mothers were not so treated (control, group C). Babies were studied twice: on days 2–5 (study 1. all infants) and days 6–10 (study 2, six infants in each group). In study 1, absolute and fractional Na⁺ excretion were significantly lower (P<0.01) and urinary K⁺Na⁺ ratio significantly higher (P<0.025) in group D than in group C, while C_cr did not differ between groups. In study 2, C_cr in group D had increased compared both with values obtained in the same babies in study 1 (P<0.05) and with group C babies in study 2 (P<0.05), but significant differences between groups in urinary Na⁺ excretion and urinary K⁺Na⁺ ratio were no longer found. We conclude that exogenous glucocorticoids accelerate maturation of renal function in immature human infants, probably by inducing tubular Na⁺. K⁺-ATPase activity. Our findings support the view that endogenous glucocorticoid hormones may play an important part in the normal maturation process.     

Digoxin- and Monensin-induced Changes of Intracellular Ca2+ Concentration in Isolated Guinea-pig Ventricular Myocyte

This study was undertaken to determine the possible mechanisms of actions of monensin and digoxin by using isolated guinea-pig ventricular myocytes. Since Ca²⁺ is the major signal for triggering contraction of cardiac muscle, the objective of this study was to determine whether monensin and digoxin affect the [Ca²⁺]_i of cardiac myocytes and if so is this effect due to an increase in [Na⁺]_i. Three different concentrations of digoxin (0.3, 1 and 3 μmol/l) and three different concentrations of monensin (0.3, 1 and 3 μmol/l) were used. Each treatment was monitored for two hours by using computerized fluoroscopy. Both digoxin and monensin increased the [Ca²⁺]_i and accelerated the onset time of [Ca²⁺]_i increase in a dose-dependent manner. Normal myocytes (loaded with fura-2 for 30 min before the treatment) were also compared with `weakened' myocytes (loaded with fura-2 for 3 h before the treatment to create a `weakened' condition). It was found that although 0.3 μmol/l monensin and digoxin did not change the [Ca²⁺]_i in normal myocytes, they increased the [Ca²⁺]_i in `weakened' myocytes. Finally, a Na<sup>+</sup>-free medium was used to demonstrate the effect of [Na<sup>+</sup>]<sub>o</sub> on both monensin- and digoxin-induced increases in [Ca<sup>2+</sup>]<sub>i</sub>. It was found that digoxin did not increase the [Ca<sup>2+</sup>]<sub>i</sub> in the Na<sup>+</sup>-free medium. Although monensin increased the [Ca<sup>2+</sup>]<sub>i</sub> in the Na<sup>+</sup>-free solution, this increase was not as large as in the Na<sup>+</sup>-containing medium. The results of the study led to the conclusion that the positive inotropic effect of digoxin depends on [Na<sup>+</sup>]<sub>o</sub>. However, monensin increases [Ca<sup>2+</sup>]<sub>i</sub> in Na<sup>+</sup>-dependent and -independent ways. An addition conclusion was that `weakened' myocytes are more sensitive to the monensin and digoxin treatment than normal myocytes.     

Purinergic Inhibition of ENaC Produces Aldosterone Escape

The mechanisms underlying “aldosterone escape,” which refers to the excretion of sodium (Na⁺) during high Na⁺ intake despite inappropriately increased levels of mineralocorticoids, are incompletely understood. Because local purinergic tone in the aldosterone-sensitive distal nephron downregulates epithelial Na⁺ channel (ENaC) activity, we tested whether this mechanism mediates aldosterone escape. Here, urinary ATP concentration increased with dietary Na⁺ intake in mice. Physiologic concentrations of ATP decreased ENaC activity in a dosage-dependent manner. P2Y₂^−/− mice, which lack the purinergic receptor, had significantly less increased Na⁺ excretion than wild-type mice in response to high-Na⁺ intake. Exogenous deoxycorticosterone acetate and deletion of the P2Y₂ receptor each modestly increased the resistance of ENaC to changes in Na⁺ intake; together, they markedly increased resistance. Under the latter condition, ENaC could not respond to changes in Na⁺ intake. In contrast, as a result of aldosterone escape, wild-type mice had increased Na⁺ excretion in response to high-Na⁺ intake regardless of the presence of high deoxycorticosterone acetate. These data suggest that control of ENaC by purinergic signaling is necessary for aldosterone escape.Renal sodium (Na⁺) excretion influences BP by affecting systemic Na⁺ balance. Consequently, negative feedback in response to changes in Na⁺ balance, perceived as changes in effective circulating volume (ECV) and BP, control renal Na⁺ handling. The fine-tuning of Na⁺ balance occurs in the aldosterone-sensitive distal nephron (ASDN), including the connecting tubule (CNT) and the collecting duct (CD). Here, Na⁺ reabsorption is highest when ECV and BP are low. Activity of the epithelial Na⁺ channel (ENaC) is limiting for discretionary Na⁺ reabsorption across the ASDN.^1–7 As ECV declines, activity of the renin-angiotensin-aldosterone system (RAAS) increases with the mineralocorticoid aldosterone, stimulating channel activity to decrease Na⁺ excretion in correction of falling ECV. Aldosterone increases the number and activity of ENaC in the apical membrane.^8–11 Under normal conditions, the opposite is also true; aldosterone and, thus, ENaC activity decline in response to elevations in BP.The thiazide-sensitive Na-Cl co-transporter (NCC) in the distal convoluted tubule (DCT) is also a target of aldosterone with the mineralocorticoid increasing NCC activity and possibly transporter abundance in the apical membrane of DCT cells.^12–16 This increase in NCC activity decreases Na⁺ excretion. Thus, aldosterone promotes distal nephron Na⁺ reabsorption by increasing the activity of ENaC and NCC.The loss of negative-feedback regulation of renal Na⁺ excretion mediated by ENaC and NCC leads to hypertension. For instance, gain-of-function mutations in ENaC cause the channel to be hyperactive irrespective of mineralocorticoid status and systemic Na⁺ balance, thus leading to inappropriate Na⁺ retention and hypertension; conversely, loss-of-function mutations in ENaC can cause decreases in BP and renal salt wasting.^{2,3,5,7,17–19}The aldosterone-promoted decrease in renal Na⁺ excretion is overridden in some circumstances.^20,21 This disruption of aldosterone action is termed aldosterone escape and results in avid Na⁺ excretion during high Na⁺ intake despite elevated mineralocorticoid levels. Aldosterone escape seems to be a protective mechanism to allow appropriate response to positive Na⁺ balance despite inappropriate mineralocorticoid levels. Such escape is important clinically, for example, in primary aldosteronism, in which it may ameliorate, to some degree, the hypertensive effects of high circulating levels of aldosterone.^22–24 The mechanism allowing aldosterone escape is uncertain.Aldosterone escape is known to be dependent on increases in renal vascular perfusion pressure rather than systemic factors, including changes in the levels of circulating hormones, such as renin and aldosterone, or renal nerve activity.²⁵ This led to the idea that aldosterone escape is a manifestation of a pressure natriuresis response.^13,25,26 This is associated with declining Na⁺ reabsorption in the distal nephron despite elevated levels of aldosterone and occurs independent of changes in GFR.^26,27 Indeed, early micropuncture measurements demonstrated increases in urine flow and Na⁺ delivery to the CD during escape.^26,27 Details about the specific site(s) along the distal nephron involved in decreased Na⁺ reabsorption during aldosterone escape and the exact transport proteins involved in this escape, though, largely remain obscure.A study by Wang et al.¹³ revealed that during aldosterone escape, NCC levels in the DCT decrease. This response was selective because the levels of other apical membrane transport proteins, including ENaC, were unaffected. This finding supports the idea that NCC is, at least, one target for regulatory processes that mediate aldosterone escape, whereby decreasing NCC abundance facilitates Na⁺ excretion despite high levels of mineralocorticoid. The cellular mechanism underpinning declines in NCC levels during aldosterone escape is unknown. Similarly, it is unclear whether other transport proteins are involved in aldosterone escape because Na⁺ reabsorption is a manifestation not only of the number of transport proteins in the apical membrane but also of their activity.Because ENaC is critical to aldosterone regulation of Na⁺ excretion, particularly in response to changes in Na⁺ balance, evidenced by the hypertension associated with gain of ENaC function,^3,5,7 we sought to investigate the role of this channel in aldosterone escape. We recently demonstrated that local purinergic tone in the ASDN exerts paracrine downregulation of ENaC activity specifically by affecting channel open probability.²⁸ This paracrine pathway is physiologically relevant because mice lacking the purinergic receptor P2Y₂, responsible for the bulk of paracrine regulation in this system, have facilitated Na⁺ reabsorption in the distal nephron and mild increases in BP.²⁹ Increases in NKCC2 and ENaC activity contribute to this elevated BP.¹¹ It does not seem a coincidence that aldosterone and P2Y₂ signaling target the same final effector proteins, possibly allowing one to compensate for the loss of the other. This idea suggests that purinergic regulation of ENaC may contribute to aldosterone escape, a possibility supported by another recent finding from our laboratories: Elevated BP in P2Y₂^−/− mice is not salt sensitive in the presence of normal-feedback regulation by RAAS but becomes salt sensitive and associated with inappropriately active ENaC when negative-feedback regulation by aldosterone and local purinergic signaling both are disrupted.^11,29To test the role of ENaC and its regulation by mineralocorticoids and purinergic signaling in aldosterone escape and to understand better the mechanism underpinning escape, we quantified the actions of mineralocorticoid on renal Na⁺ excretion, ENaC activity, and urinary ATP levels in wild-type (WT) and P2Y₂^−/− mice stressed with different dietary Na⁺ regimens. We found that urinary [ATP] increases with dietary Na⁺ intake such that physiologic [ATP] decrease ENaC activity, resulting in increased Na⁺ excretion. Increased Na⁺ excretion is greater in WT compared with P2Y₂^−/− mice and associated with an inability of ENaC, particularly when mineralocorticoid is clamped at high levels, to respond appropriately to changes in dietary Na⁺ intake in the latter animals. Because ENaC activity normally is sensitive to changes in Na⁺ balance even when mineralocorticoids are clamped at high levels, these results show that control of ENaC by purinergic signaling is necessary for complete aldosterone escape, consistent with the loss of aldosterone-escape in P2Y₂^−/− mice and their pronounced hypertension relative to normal mice in the presence of elevated mineralocorticoids and high Na⁺ intake.     

Sodium Transport Is Modulated by p38 Kinase–Dependent Cross-Talk between ENaC and Na,K-ATPase in Collecting Duct Principal Cells

In relation to dietary Na⁺ intake and aldosterone levels, collecting duct principal cells are exposed to large variations in Na⁺ transport. In these cells, Na⁺ crosses the apical membrane via epithelial Na⁺ channels (ENaC) and is extruded into the interstitium by Na,K-ATPase. The activity of ENaC and Na,K-ATPase must be highly coordinated to accommodate variations in Na⁺ transport and minimize fluctuations in intracellular Na⁺ concentration. We hypothesized that, independent of hormonal stimulus, cross-talk between ENaC and Na,K-ATPase coordinates Na⁺ transport across apical and basolateral membranes. By varying Na⁺ intake in aldosterone-clamped rats and overexpressing γ-ENaC or modulating apical Na⁺ availability in cultured mouse collecting duct cells, enhanced apical Na⁺ entry invariably led to increased basolateral Na,K-ATPase expression and activity. In cultured collecting duct cells, enhanced apical Na⁺ entry increased the basolateral cell surface expression of Na,K-ATPase by inhibiting p38 kinase-mediated endocytosis of Na,K-ATPase. Our results reveal a new role for p38 kinase in mediating cross-talk between apical Na⁺ entry via ENaC and its basolateral exit via Na,K-ATPase, which may allow principal cells to maintain intracellular Na⁺ concentrations within narrow limits.The fine-tuning of Na⁺ balance is critical for the homeostasis of body fluid compartments. A variety of disorders and diseases, such as hypertension and edema, result at least partly from disturbances of Na⁺ homeostasis.¹ The final regulation of renal Na⁺ reabsorption takes place in aldosterone-responsive distal tubules and collecting ducts.² The bulk of Na⁺ transport in the collecting duct (CD) occurs in principal cells, where Na⁺ enters the cell via the epithelial sodium channel (ENaC) and is extruded into the interstitial compartment via Na,K-ATPase.³ Thus, tight control of vectorial Na⁺ transport must be exerted on CD principal cells to achieve whole-body Na⁺ homeostasis.According to dietary Na⁺ intake and aldosterone levels, CD principal cells are exposed to large physiologic variations of Na⁺ transport.^2,3 Meanwhile, intracellular Na⁺ concentration must be maintained within narrow ranges, which is essential for vital cellular functions, such as control of osmolality, ionic strength, and membrane potential. Therefore, apical Na⁺ entry and basolateral Na⁺ extrusion must be rapidly and tightly coordinated in order to match variations of Na⁺ transport while minimizing fluctuations of intracellular Na⁺ concentration. The mechanisms mediating this coordination remain largely unknown.Control of exocytosis/endocytosis is a common mechanism for modulating the abundance and function of membrane proteins. For example, increasing the activity of the AMP-activated protein kinase (AMPK), as a result of increased ATP consumption, modulated Na,K-ATPase endocytosis in cultured renal epithelial MDCK cells.⁴ Among several actions, activation of p38 kinase, a member of the MAP kinase family, regulates the endocytosis of a variety of cell surface proteins.⁵ We reported previously that aldosterone treatment which stimulates active transcellular Na⁺ reabsorption reduced p38 kinase activation, but not that of ERK1/2, in renal CD principal cells.⁶ Activation of p38 kinase is essential for EGFR endocytosis and lysosomal degradation.^7–9 Interestingly, p38 kinase controls the phosphorylation and ubiquitinylation of aquaporin-2 (AQP2), triggering its endocytosis and degradation in renal CD principal cells.¹⁰We hypothesized that CD principal cells exhibit tight coordination of apical and basolateral Na⁺ transport, putatively through modulation of Na,K-ATPase cell surface expression by Na⁺ apical entry. AMPK and/or p38 kinase signaling pathways may control Na,K-ATPase endocytosis involved in cross-talk between ENaC and Na,K-ATPase. In this study, we describe a cross-talk between apical ENaC and basolateral Na,K-ATPase in a physiologic context. We identified p38 kinase-regulated endocytosis and degradation of cell surface Na,K-ATPase as a key player in this cross-talk.     

Aprikalim reduces the Na-Ca exchange outward current enhanced by hyperkalemia in rat ventricular myocytes

Bacground. Aprikalim, an adenosine triphosphate (ATP) sensitive K⁺ (K_ATP) channel opener, attenuates the elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and improves the contractile functions after hyperkalemic and hypothermic cardioplegia. There is evidence that cardioplegia increases the Na⁺-Ca²⁺ exchange activity without affecting Ca²⁺ influx through L-type Ca²⁺ channels or Ca²⁺ content in the sarcoplasmic reticulum, the intracellular Ca²⁺ store.Methods. We measured the Na⁺-Ca²⁺ exchange outward current with the patch-clamp technique in single rat ventricular myocytes exposed to hyperkalemia and hypothermia in the presence of aprikalim. The intracellular calcium concentration ([Ca²⁺]_i) during cardioplegia, and the contractile function and [Ca²⁺]_i transients induced by electrical stimulation or caffeine during rewarming and reperfusion in single ventricular myocytes were also determined. Contraction and [Ca²⁺]_i were determined with video tracking and spectrofluorometry, respectively.Results. Aprikalim, 100 μmol/L, the effect of which was blocked by glibamclamide, a K_ATP inhibitor, significantly attenuated the hyperkalemia-elevated Na⁺-Ca²⁺ exchange current by 26% and 11% at 22°C and 4°C, respectively. Aprikalim also attenuated significantly the [Ca²⁺]_i elevated during cardioplegia. Furthermore aprikalim significantly attenuated the reduction in amplitude and prolongation in duration of contraction of myocytes after cardioplegia. The effects of aprikalim mimicked those of nickle (Ni²⁺), a Na⁺-Ca²⁺ exchange blocker. The electrically or caffeine-induced [Ca²⁺]_i transients were unaltered by cardioplegia or aprikalim.Conclusions. Aprikalim attenuates the Na⁺-Ca²⁺ exchange outward current elevated by hyperkalemia, which may attenuate the [Ca²⁺]_i elevation during hyperkalemia and improve the contractile function after cardioplegia in the ventricular myocyte. The study provides further support that addition of a K_ATP channel opener to the cardioplegic solution may produce beneficial effects in open heart surgery.     

Transdermal Delivery of Chloroquine by Amidated Pectin Hydrogel Matrix Patch in the Rat

The aim of the present study was to investigate the suitability of amidated pectin matrix patch for transdermal chloroquine delivery in an effort to mask the bitter taste when orally administered. Chloroquine has easily measurable outputs that are linked to increased renal Na⁺ excretion. We thus monitored urinary Na⁺ output in separate groups intravenously administered chloroquine or topically applied pectin hydrogel chloroquine matrix patch. Male groups of anesthetized Sprague-Dawley rats were placed on a continuous jugular infusion of 0.077 M NaCl at 150 µL min^?1. After 3 h equilibration period, consecutive 20 min urine collections were made over the subsequent 4 h of 1 h control, 1 h 20 min treatment, and 1 h 40 min recovery periods for measurements of urine flow and Na⁺ and K⁺ excretion rates. The effects of intravenous chloroquine infusion or topical application of pectin hydrogel chloroquine matrix patch were examined in rats in which the drug was added to the infusate or patch applied onto the shaved area during the 1 h 20 min treatment period. The animals were switched back to the infusate alone for the final 1 h 40 min recovery period. Vehicle infused animals acted as controls. Trunk blood was collected after the treatment period from parallel groups for chloroquine measurements. The plasma chloroquine concentrations following iv chloroquine or application of pectin chloroquine hydrogel matrix patch were 9.3 ± 0.8 mg L^?1 and 7.3 ± 1.1 mg L^?1, respectively (n = 7 in both groups). Chloroquine infusion and pectin chloroquine patch significantly (p<0.01) increased Na⁺ excretion to peak values of 14.1 ± 0.9 µmol min^?1, and 20.35 ± 1.0 µmol min^?1, respectively by comparison with controls (9.1 ± 0.9 µmol min^?1), at the corresponding period. The results suggest that the pectin chloroquine patch matrix preparation has potential applications for transdermal delivery of chloroquine and perhaps in the management of malaria.     

Differentiation of proton-pumping activity in cultured renal inner medullary collecting duct cells

Cultured inner medullary collecting duct (IMCD) cells have been shown to secrete protons (H⁺) by two mechanisms: anN-ethylmaleimide-and dicyclohexyl-carbodiimide-sensitive electrogenic H⁺-ATPase or H⁺ pump, and an amiloride-sensitive, secondary active Na⁺/H⁺ exchanger. These cells also express Cl^–/HCO₃ ^– exchange and carbonic anhydrase activity in common with other renal epithelial cells involved in acid-base transport. Video fluorescence microscopy of individual cells using 2, 7-biscarboxyethyl-5(6)-carboxyfluorescein has demonstrated that adjacent-cultured IMCD cells show substantial functional intercellular heterogeneity. The development of H⁺-pumping activity is associated with high-baseline intracellular pH and peanut agglutinin (PNA) affinity, and loss of mitotic activity and of Na⁺/H⁺ exchange. The H⁺-pumping activity may be further enhanced by removal of fetal calf serum for 6–54 h or by selecting cells with high PNA affinity. IMCD cells in their most differentiated state form domes, which consistently showed the highest rates of H⁺-pumping activity, as well as high affinity for peanut lectin. When IMCD were plated at low density, domes developed relatively late (2–4 weeks), at which time cells located in the center of nests of contiguously growing cells were quiescent and showed H⁺-pumping activity but no Na⁺/H⁺ exchange. On the other hand, dense plating was associated with early development of domes (end of 1st week), at which time adjacent cells showed a high mitotic activity and Na⁺/H⁺ exchange, but no H⁺-pumping activity. We speculate that differentiation of IMCD cells results in the development of cell polarity. This could include either loss of the apical Na⁺/H⁺-exchange activity, or localization of this exchanger only to the basolateral membrane, while the H⁺ pump differentiates at the apical membrane.     

High- or low-potassium solutions for the storage of abdominal and thoracic organs

Background: Clinically, intracellular type solutions are the most widely used solutions to preserve organs. The optimal ion composition of preservation solutions, however, is still unknown and extracellular-type solutions have frequently been superior to intracellular solutions in various experimental studies. Materials and methods: In this study, we measured extracellular (interstitial) electrolyte concentrations in rat livers, kidneys, hearts and lungs at 4°C by means of microdialysis sampling. Results: After 24 h cold ischaemia, [Na⁺]_int and [K⁺]_int were 104±25 mmol/l and 6.5±0.7 mmol/l in hearts, 92±12 mmol/l and 6.9±1.0 mmol/l in livers, 115±22 mmol/l and 6.3±0.9 mmol/l in kidneys and 87±17 mmol/l and 6.4±0.6 mmol/l in lungs. After preservation of organs in intracellular-type solutions, [Na⁺]_int was significantly lower for each organ (range from 69±8 mmol/l to 73±20 mmol/l) and [K⁺]_int was significantly higher (range from 8.0±1.7 mmol/l to 9.8±1.0 mmol/l). In no instance did the interstitial electrolyte concentration equilibrate with the intracellular electrolyte concentration. When the diffusion gradient from the vascular space to the interstitial space was calculated for Na⁺ and K⁺, a significantly higher barrier was found for K⁺ than for Na⁺ (P<0.001 and P<0.01 for hearts). Conclusions: These studies indicate that during cold storage of rat hearts, lungs, livers and kidneys, intra- and extracellular electrolytes do not equilibrate. Ion exchange stabilises at extracellular Na⁺ concentrations between 87 mmol/l and 115 mmol/l and K⁺ concentrations between 6.3 mmol/l and 6.9 mmol/l. Storage of organs in solutions with extracellular-type ion compositions might improve graft function and survival not only after lung and liver but also after heart and renal preservation. Received: 4 August 1999 In revised form: 23 February 2000 Accepted: 24 February 2000     

The renal transport of taurine and the regulation of renal sodium-chloride-dependent transporter activity

A model for the -amino acid taurine transport is presented to help define the ionic, pH, and voltage requirements for the movement of taurine into the rat proximal tubule brush border membrane vesicle (BBMV). Sodium-(Na⁺)-taurine symport across the apical surface of the proximal tubule has a highly specific requirement for Cl^– and Br^–. Active taurine transport operates with a 2 Na⁺: 1 Cl^–: 1 taurine-carriier complex. Complexes like the one required for maximal taurine transport may be pertinent for many other amino acids whose uptake is Na⁺-dependent. Renal epithelial cell lines LLC-PK and MDCK were used to define the nature of taurine uptake; they express Na⁺-Cl^–-taurine cotransport that is inhibited by -alanine. The cell lines up-or down-regulate taurine transport in response to changes in the taurine concentration of the medium in a manner similar to that seen in BBMV. The adaptation is present by 12 h and depends on new protein synthesis and protein import to the cell membrane. The role of trafficking in the adaptive response was also explored in brush border vesicles. During dietary surfeit, transporter could be down-regulated and transporters could be shifted back into the microtubule system, resulting in taurinuria. Use of continuous renal cell lines allowed a more mechanistic exploration of intracellular trafficking in the up- and down-expression of the Na⁺-Cl^–-taurine cotransporter. Colchicine appeared to be a more potent inhibitor of the rapid (over hours) adaptive response to a reduction in media and, therefore, intracellular taurine content. This effect suggests that the increase in Na⁺ symporter is due to import of a transport protein into functional sites in the brush border membrane, rather than a major increase in mRNA synthesis. The proximal tubule luminal membrane of the neonatal rat is characterized by a high amiloride-sensitive Na⁺/H⁺ exchange activity and high permeability to Na⁺. The resulting increase in Na⁺ transport in the immature proximal tubule may contribute to the positive Na⁺ balance of the growing organism and may potentially influence the pattern of urinary solute excretion during early life, including an increase in urinary taurine excretion.     

Calcium-sensing receptor (CaSR) modulates vacuolar H<Superscript>+</Superscript>-ATPase activity in a cell model of proximal tubule

Background

The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H⁺-ATPase in a cellular model of proximal tubule cells, OKP cells.

Methods

Biochemical activity of H⁺-ATPase was performed using cell homogenates, and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium [Ca²⁺]_i were also determined using Fluo-4.

Results

A significant increase of vacuolar H⁺-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd³⁺ (300 µM) and neomycin (200 µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca²⁺ (Ca²⁺_o) between 10^?4 and 2 mM. Gd³⁺ and neomycin produced a sustained rise of [Ca²⁺]_i, an effect that disappears when extracellular calcium was removed in the presence of 0.1 µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5?×?10^?8 M) reduced the increase in [Ca²⁺]_i induced by neomycin.

Conclusion

CaSR stimulation induces an increase in the vacuolar H⁺-ATPase activity of OKP cells, an effect that involves an increase in [Ca²⁺]_i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

     

Ca2+ uptake by endoplasmic reticulum of renal cortex. I. Ionic requirements and regulation in vitro

Summary A subcellular fraction enriched in cytochrome c reductase (7.9-fold) and relatively de-enriched (0.64-fold) in Na⁺/K⁺-ATPase was prepared from canine kidney cortex by sucrose density gradient ultracentrifugation. It was shown by electron microscopy to consist primarily of a light fraction of endoplasmic reticulum (LER). LER vesicles displayed ATP-dependent ⁴⁵Ca²⁺ uptake that was insensitive to 10 mM KCN or NaN₃, and was promptly released by 20 M A23187 or ionomycin. Inositol-1,4,5-trisphosphate (IP₃) appeared to produce a time-dependent release of ⁴⁵Ca²⁺. Vanadate inhibited ⁴⁵Ca²⁺ uptake with a K_i0.3 mM, further suggesting that the activity resided in the ER rather than the plasma membrane. ⁴⁵Ca²⁺ uptake by LER, at 5 M total [Ca²⁺], displayed a strong dependence on divalent cations (Mg²⁺>Co²⁺>Mn²⁺Ba²⁺Cd²⁺Sr²⁺, present at 2 mM) as well as on monovalent cations (Na⁺K⁺+Na⁺ >K⁺>Li⁺>choline⁺), and anions (Cl^->acetate^-NO₃ ^-F^->H₂PO₄ ^-gluconate^-oxalate⁼SO₄ ⁼). It had a fairly narrow pH optimum (7.25–7.50). Preincubation (10 min) of LER vesicles with 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated LER Ca²⁺ uptake; this effect was enhanced in the presence of renal cytosol [5% (vol/vol)]. However, Ca²⁺ uptake was not affected by preincubation with dibutyryl cyclic AMP, calmodulin, or 1,25-(OH)₂-vitamin D₃, either in the absence or presence of renal cytosol. Thus, the Mg²⁺-ATP dependent ⁴⁵Ca²⁺ uptake activity of this canine renal cortical LER fraction displays modulation by IP₃, TPA, and pH that appears to be physiologically relevant. Offprint request to}: D. Moskowits, Nephrology Division 111B-JC Montreal Canada     

A comparative study of the exchangein vivo of major constituents of bone mineral

Groups of young and old rats were injected with a variety of labelled substanzes (urea, Cl^–, K⁺, Na⁺, HCO ₃ ^– , PO ₄ ^3– , Ca⁺⁺). Data for Mg⁺⁺ were taken from the literature. One and a half hours later, compact shafts of long bones were removed and cleaned scrupulously, and analyses were performed for both cold and isotopic concentrations of substances. This time point was chosen to insure equilibration of the aqueous phase of bone while minimizing contributions from surface exchange, recrystallization, solid diffusion, growth or resorption.With fixed variables of time, species, bone specimen, and methodology, uambiguous comparisons of the exchange in bone could be made between the many substances studied. The exchange data could be divided into three categories: a) complete exchange (urea Cl^–, and K⁺); b) partial exchange, decreasing variably with age (Na⁺, CO₂, and Mg⁺⁺); and c) minimal exchange (Ca⁺⁺ and PO ₄ ^3– ). Clearly the traditional classification of available and unavailable skeleton is ambiguous and determined by the conditions and the ion or substance chosen for study. Clearly also, a new overall concept of bone exchangein vivo is badly needed.Calculations of the apparent concentration of the various electrolytes in bone water reveal that the aqueous phase of bone has a composition markedly different from plasma water. In particular, the concentration of potassium in bone water was found to be remarkably high.This work was supported in part by the United States Public Health Service grants no. 1 T1 DE 175 and R-501-Am 08271 and in part by the United States Atomic Energy Commission, contract no. W-7401-Eng-49 and has been assigned Report No. UR-49-898.     

Effect of Poloxamer 188 on deepening of deep second-degree burn wounds in the early stage

Objective

To discuss the effect of Poloxamer 188 (P188) on deepening of deep second-degree burn wounds in the early stage after burn.

Methods

We divided Wistar rats with deep second-degree burn wounds on the backs thereof into two groups, then intravenously injected P188 for the treatment group and intravenously injecting physiological saline for the control group, detecting the activity of Na⁺–K⁺–adenosine triphosphatase (Na⁺–K⁺–ATPase), myeloperoxidase (MPO) and the content of malonaldehyde (MDA) and succinic dehydrogenase (SDH) in the burn wound, and showing the degree of necrosis in the wound by haematoxylin–eosin (HE) and proliferating cell nuclear antigen (PCNA) immunohistochemical staining.

Results

In the control group and treatment group, the activity of SDH and Na⁺–K⁺–ATPase dropped to the lowest point 24 h after the burn took place, and then increased gradually, but was still far lower than the normal level at the furthest time point. At 24 h after burn, activity of SDH and Na⁺–K⁺–ATPase in the treatment group was higher than that of the control group (P < 0.05); the activity of MPO of the control group reached the highest point at 24 h while that of MPO of the treatment group reached the highest point after 48 h; later, that of MPO of both groups decreased, but was still higher than the normal level. Compared with the highest values of the activity of MPO of both groups, that of the control group was higher than that of the treatment group (p < 0.05); the contents of MDA of both groups kept increasing after the burn; 72 h later, that of the control group was higher than that of the treatment group (p < 0.05). HE and PCNA staining showed progressive damage of the wound in the treatment group, which was decreased with treatment, particularly at the early stages.

Conclusion

Systemic application of P188 on deep second-degree burn wounds at the early stage may alleviate wound deepening, whose mechanism may be related to timely sealing up the damaged cell membrane and inhibiting the inflammatory reaction.     

Hypoxia upregulates amino acid transport in a human neuroblastoma cell line

Background/Purpose

Some tumor cells survive and even grow in hypoxic conditions. We examined the effect of hypoxia on amino acid transport in a human neuroblastoma cell line SK-N-SH.

Methods

Cells were incubated under hypoxic conditions (1% O₂-5% CO₂-94% N₂). After 0, 8, 16, and 24 hours, the transport of ³H-glutamine, ³H-glutamate, and ³H-leucine was assayed. ³H-Thymidine and ³H-leucine incorporation was measured for the determination of DNA and protein synthesis, respectively.

Results

Hypoxia increased Na⁺-dependent glutamine and Na⁺-independent leucine transport significantly at 16 and 24 hours compared with control (P < .01) by a mechanism that increased V_max without affecting K_m. These increases were completely blocked by actinomycin D and cycloheximide. There was no significant difference in Na⁺-dependent glutamate transport between control and hypoxic groups. DNA and protein synthesis significantly decreased in the hypoxic condition compared with control (P < .01).

Conclusions

This study demonstrated that hypoxia upregulates amino acid transport in a human neuroblastoma cell line. This mechanism may allow cells to survive and even grow under hypoxic conditions.