Blunted DOCA/high salt induced albuminuria and renal tubulointerstitial damage in gene-targeted mice lacking SGK1

Mineralocorticoids stimulate renal tubular Na⁺ reabsorption, enhance salt appetite, increase blood pressure, and favor the development of renal fibrosis. The effects of mineralocorticoids on renal tubular Na⁺ reabsorption and salt appetite involve the serum- and glucocorticoid-inducible kinase 1 (SGK1). The kinase is highly expressed in fibrosing tissue. The present experiments thus explored the involvement of SGK1 in renal fibrosis. To this end, SGK1-knockout mice (sgk1 ^−/−) and their wild-type littermates (sgk1 ^+/+) were implanted with desoxycorticosterone acetate (DOCA)-release pellets and offered 1% saline as drinking water for 12 weeks. The treatment led to significant increases in fluid and Na⁺ intake and urinary output of fluid and Na⁺ in sgk1 ^+/+ mice, effects blunted in sgk1 ^−/− mice. Blood pressure increased within the first 7 weeks to a similar extent in both genotypes, but within the next 5 weeks, it increased further only in sgk1 ^+/+ mice. Creatinine clearance did not change significantly but albuminuria increased dramatically in sgk1 ^+/+ mice, an effect significantly blunted in sgk1 ^−/− mice. Histology after 12 weeks treatment revealed marked glomerular sclerosis and tubulointerstitial damage with interstitial fibrosis and inflammation in kidneys from sgk1 ^+/+ mice, but not from sgk1 ^−/− mice. In conclusion, a lack of SGK1 protects against DOCA/high-salt-induced albuminuria and renal fibrosis.     

SGK1 dependence of insulin induced hypokalemia

Insulin stimulates cellular K⁺ uptake leading to hypokalemia. Cellular K⁺ uptake is accomplished by parallel stimulation of Na⁺/H⁺ exchange, Na⁺,K⁺,2Cl⁻ co-transport, and Na⁺/K⁺ ATPase and leads to cell swelling, a prerequisite for several metabolic effects of the hormone. Little is known about underlying signaling. Insulin is known to activate the serum and glucocorticoid-inducible kinase SGK1, which in turn enhances the activity of all three transport proteins. The present study thus explored the contribution of SGK1 to insulin-induced hypokalemia. To this end, gene-targeted mice lacking SGK1 (sgk1 ^−/− ) and their wild-type littermates (sgk1 ^+/+ ) have been infused with insulin (2 mU kg⁻¹ min⁻¹) and glucose at rates leaving the plasma glucose concentration constant. Moreover, isolated liver perfusion experiments have been performed to determine stimulation of cellular K⁺ uptake by insulin (100 nM). As a result, combined glucose and insulin infusion significantly decreased plasma K⁺ concentration despite a significant decrease of urinary K⁺ excretion in sgk1 ^+/+ but not in sgk1 ^−/− mice. Accordingly, the plasma K⁺ concentration was within 60 min significantly lower in sgk1 ^+/+ than in sgk1 ^−/− mice. In isolated liver perfusion experiments, cellular K⁺ uptake was stimulated by insulin (100 nM), an effect blunted by 72% in sgk1 ^−/− mice as compared to sgk1 ^+/+ mice. Accordingly, insulin-induced cell hydration was 63% lower in sgk1 ^−/− mice than in sgk1 ^+/+ mice. Moreover, volume regulatory K⁺ release was 31% smaller in sgk1 ^−/− mice than in sgk1 ^+/+ mice. In conclusion, the serum and glucocorticoid-inducible kinase SGK1 participates in the signaling mediating the hypokalemic effect of insulin.     

Renal Ca2+ handling in sgk1 knockout mice

Coexpression studies in Xenopus oocytes revealed the ability of the serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate the renal epithelial Ca²⁺ channel TRPV5. SGK1 increases the abundance of the channel protein in the plasma membrane, an effect requiring the participation of the Na⁺/H⁺ exchanger regulating factor 2 (NHERF2). The present study was performed to explore the role of SGK1 in the regulation of renal Ca²⁺ handling in vivo. To this end, TRPV5, calbindin D-28K abundance, and renal Ca²⁺ excretion were analyzed in gene-targeted mice lacking functional SGK1 (sgk1 ^−/− ) and their age- and sex-matched littermates (sgk1 ^+/+ ). Immunohistochemistry revealed lower abundance of TRPV5 and calbindin D-28K protein in sgk1 ^−/− mice than in sgk1 ^+/+ mice, both fed with control diet. Feeding the mice a Ca²⁺-deficient diet marked ly increased TRPV5 protein abundance in both genotypes. Renal Ca²⁺ excretion under control diet was significantly lower in sgk1 ^−/− than in sgk1 ^+/+ mice. The Ca²⁺-deficient diet decreased renal excretion of Ca²⁺ to the same levels in both phenotypes. Furosemide increased fractional Ca²⁺ excretion and dissipated the difference between phenotypes. We conclude that lack of SGK1 may lead to decrease in TRPV5 abundance in connecting tubules but does not abrogate TRPV5 regulation. The decrease in abundance of TRPV5 in connecting tubules of sgk1 ^−/− mice is presumably compensated for by enhanced Ca²⁺ reabsorption in upstream nephron segments such as the loop of Henle, which may indirectly result from impaired SGK1-dependent Na⁺ reabsorption in the aldosterone-sensitive distal part of the nephron, salt loss, and enhanced Na⁺ (and Ca²⁺) reabsorption in those upstream nephron segments.D. Sandulace and F. Grahammer contributed equally to this work.     

Decreased intestinal glucose transport in the sgk3-knockout mouse

Xenopus oocyte coexpression experiments revealed the capacity of the serum- and glucocorticoid-inducible kinase isoform 3 (SGK3) to up-regulate a variety of transport systems including the sodium-dependent glucose transporter SGLT1. The present study explored the functional significance of SGK3-dependent regulation of intestinal transport. To this end, experiments were performed in gene targeted mice lacking functional sgk3 (sgk3^–/–) and their wild type littermates (sgk3^+/+). Oral food intake and fecal dry weight were significantly larger in sgk3^–/– than in sgk3^+/+ mice. Glucose-induced current (I_g) in Ussing chamber as a measure of Na⁺ coupled glucose transport was significantly smaller in sgk3^–/– than in sgk3^+/+ mouse jejunal segments. Fasting plasma glucose concentrations were significantly lower in sgk3^–/– than in sgk3^+/+ mice. Intestinal electrogenic transport of phenylalanine, cysteine, glutamine and proline were not significantly different between sgk3^–/– and sgk3^+/+ mice. In conclusion, SGK3 is required for adequate intestinal Na⁺ coupled glucose transport and impaired glucose absorption may contribute to delayed growth and decreased plasma glucose concentrations of SGK3 deficient mice. The hypoglycemia might lead to enhanced food intake to compensate for impaired intestinal absorption.Ciprian Sandu, Rexhep Rexhepaj Shared first authorship.     

SGK1-dependent salt appetite in pregnant mice

Aim: Pregnancy is typically paralleled by substantial increase in maternal extracellular fluid volume, requiring net accumulation of water and NaCl. The positive water and salt balance is accomplished at least in part by increased uptake of salt secondary to enhanced salt appetite. Little is known about the underlying cellular mechanisms. Stimulation of salt appetite by mineralocorticoids, however, is known to be dependent on the serum‐ and glucocorticoid‐inducible kinase SGK1. Methods: To test for a role of SGK1 in the stimulation of salt appetite during pregnancy, fluid intake was recorded in pregnant SGK1 knockout mice (sgk1^?/?) and their wild type littermates (sgk1^+/+). The mice were offered two bottles, one with plain water and the other with isotonic saline. Results: In early pregnancy, i.e. up to 10 days prior to parturition, the sgk1^+/+ mice displayed a significant preference for saline, whereas the sgk1^?/? mice preferred water. Accordingly, the water intake was significantly smaller and saline intake was significantly larger in sgk1^+/+ mice than in sgk1^?/? mice and the preference for water was significantly stronger in sgk1^?/? mice than in sgk1^+/+ mice. Plasma aldosterone levels were higher in sgk1^?/? mice than in sgk1^+/+ mice, a difference contrasting the enhanced salt appetite of sgk1^+/+ mice. Conclusions: SGK1 participates in the stimulation of salt appetite during pregnancy.     

Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

Stimulation of electrogenic intestinal dipeptide transport by the glucocorticoid dexamethasone

According to recent in vitro experiments, the peptide transporter PepT2 is stimulated by the serum- and glucocorticoid-inducible kinase SGK1. The present study explored the contribution of SGK1 to the regulation of electrogenic intestinal peptide transport. Intestinal PepT1 was expressed in Xenopus oocytes, and peptide transport was determined by dual electrode voltage clamping. Peptide transport in intestinal segments was determined utilizing Ussing chamber. Cytosolic pH (pH _i ) was determined by BCECF fluorescence and Na⁺/H⁺ exchanger activity was estimated from Na⁺-dependent pH recovery (?pH _i ) following an ammonium pulse. In PepT1-expressing Xenopus oocytes, coexpression of SGK1 enhanced electrogenic peptide transport. Intestinal transport and pH _i of untreated mice were similar in SGK1 knockout mice (sgk1 ^?/? ) and their wild-type littermates (sgk1 ^+/+ ). Glucocorticoid treatment (4 days 10 µg/g body weight (bw)/day dexamethasone) increased peptide transport in sgk1 ^+/+ but not in sgk1 ^?/? mice. Irrespective of dexamethasone treatment, luminal peptide (5 mM glycyl-glycine) led to a similar early decrease of pH _i in sgk1 ^?/? and sgk1 ^+/+ mice, but to a more profound and sustained decline of pH _i in sgk1 ^?/? than in sgk1 ^+/+ mice. In the presence and absence of glycyl-glycine, ?pH _i was significantly enhanced by dexamethasone treatment in sgk1 ^+/+ mice, an effect significantly blunted in sgk1 ^?/? mice. During sustained exposure to glycyl-glycine, ?pH _i was significantly larger in sgk1 ^+/+ mice than in sgk1 ^?/? mice, irrespective of dexamethasone treatment. In conclusion, basal intestinal peptide transport does not require stimulation by SGK1. Glucocorticoid treatment stimulates both Na⁺/H⁺ exchanger activity and peptide transport, effects partially dependent on SGK1. Moreover, chronic exposure to glycyl-glycine stimulates Na⁺/H⁺ exchanger activity, an effect again involving SGK1.     

Effects of antidiuretic hormone,parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP, 10⁻¹⁰mol.l⁻¹), parathyroid hormone (PTH, 10⁻⁸ mol.l⁻⁸) and glucagon (10⁻⁸ mol.l⁻¹) on the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were tested in in vitro perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. When compared with mTAL segments (PD_te: 8.5±0.4 mV,n=16), cTAL segments displayed a high PD_te of 15.7±0.9 mV (n=11) at the beginning of perfusion experiments which reached a value of 9.4±0.6 mV (n =11) after 38±4 min perfusion. Simultaneously R_te increased significantly from 24±3 to 28±1 Ω cm² (n=11). When PTH, AVP or glucagon were added to the bath solution, PD_te increased with PTH from 10.3±0.8 to 15.2±0.8 mV (n=13), with AVP from 10.2±0.5 to 15.0±0.7 mV (n=24) and with glucagon from 11.3±1.9 to 15.3±2.1 mV (n=8). At the same time R_te decreased from 30±3 to 23±2 Ω cm², from 28±1 to 23±1 Ω cm² and from 23±2 to 18±2 Ω cm², respectively. In mTAL segments, AVP and glucagon increased PD_te from 8.4+0.5 to 13.5±0.9 mV (n=11) and from 8.8±0.6 to 12.8±0.6 mV (n=8) respectively, while R_te decreased significantly from 23±1 to 20±1 Ω cm² and from 27±3 to 21±3 Ω cm². PTH, on the other hand, had no effect on PD_te and R_te. Since the response to PTH appeared to be specific to cTAL segments, paired experiments were performed, in which AVP or glucagon were successively tested with PTH on cTAL and mTAL segments, to ascertain the specificity of the hormonal response. In cTAL segments, PTH and AVP increased the equivalent short-circuit current (I_sc=PD_te/R_te) by 82% and 86% respectively, while PTH and glucagon, in another series, increased I_sc by 95% and 81% respectively. In mTAL segments, I_sc was increased in the presence of AVP and glucagon by 88%, and 93% respectively, whereas PTH had no effect. These results indicate that Nacl reabsorption in cTAL segments is stimulated by AVP, PTH and glucagon and in mTAL segments by AVP and glucagon. The amplitude of the response to the hormones is similar in the two segments. The residual stimulation in cTAL segments, however, persists longer than in mTAL segments.     

Potential contribution of vasoconstriction to suppression of heat loss and homeothermic regulation in UCP1-deficient mice

To investigate the thermoregulatory mechanism in mice lacking uncoupling protein 1 (UCP1) from the viewpoint of heat loss, we measured oxygen consumptions (VO2), skin-surface temperatures (Tskin, an index of heat release), blood flows in the tails, and rectal temperatures (Trectal) of mice housed in an animal room under the standard thermal condition of ∼23°C. Compared with wild-type (Ucp1 ^+/+) mice, adult UCP1-deficient (Ucp1 ^−/−) mice tended to show a reduced VO2. Thermograhic analysis of the acute response of Ucp1 ^−/− mice to a small change (a drop of 1–2°C) in the ambient temperature revealed a sustained fall in the Tskin of Ucp1 ^−/− mice; but this fall was only transient in Ucp1 ^+/+ mice. Analysis of tail blood flow under anesthesia clearly showed a stronger vasoconstrictor response in Ucp1 ^−/− mice than in Ucp1 ^+/+ mice. Administration of a vasodilator, evodiamine, transiently increased Tskin in Ucp1 ^+/+ and Ucp1 ^−/− mice similarly; whereas the induction of vasodilation caused a greater and more prolonged reduction in Trectal in Ucp1 ^−/− mice than in Ucp1 ^+/+ mice. These results indicate that Ucp1 ^−/− mice highly, or at least partly, rely on vasoconstriction for heat conservation to compensate for their UCP1 deficiency and to maintain homeothermy under the condition of normal housing temperature.     

Vasopressin stimulation of NaCl transport in the medullary thick ascending limb of Henle's loop is decreased in aging mice

The maximal urinary osmolality that can be reached by the kidney is reduced with age. This may be due to impaired NaCl transport by the medullary thick ascending limb of Henle's loop, which is part of the renal concentrating mechanism and is modulated by antidiuretic hormone (ADH). We therefore tested in vitro a possible age-related change in the transport capacity and in the response of this nephron segment to ADH in young (1–2 months) and old (20–24 months) mice. The transepithelial potential difference (V _te) was significantly higher in young mice (+8.5±0.4 mV, n=13) than in old ones (+6.6±0.5 mV, n=17). Addition of 0.1 nmol.l^–1 ADH to the bath solution significantly increased V _te by 5.2±0.5 mV in the young and by 3.1±0.6 mV in the old animals. Application of dibutyryl-cAMP (0.1 mmol.1^–1) did not further increase the hormonal response in both groups. The ADH-mediated increase in the corresponding equivalent short-circuit current (I _SC = V _te/R_te) was twice as great in young mice as in old, indicating that the stimulation of NaCl transport by ADH across the medullary thick ascending limb is significantly reduced with age. These results suggest that the previously reported age-related defect in the urinary concentrating ability of the kidney is partly due to a decreased response of the medullary thick ascending limb to ADH.     

Amiloride-sensitive Na+ transport across cultured renal (A6) epithelium: evidence for large currents and high NaK selectivity

Electrical techniques were used to determine the NaK selectivity of the amiloride-sensitive pathway and to characterize cellular and paracellular properties of A6 epithelium. Under control conditions, the mean transepithelial voltage (V _T) was –57±5 mV, the short-circuit current (I _sc) averaged 23±2 A/cm² and the transepithelial resistance (R _T) was 2.8±0.3 kcm² (n=13). V _T and I _sc were larger than reported in previous studies and were increased by aldosterone. The conductance of the amiloride-sensitive pathway (G _amil) was assessed before and after replacement of Na⁺ in the mucosal bath by K⁺, using two independent measurements: (1) the slope conductance (G _T), determined from current-voltage (I-V) relationships for control and amiloride-treated tissues and (2) the maximum amiloride-sensitive conductance (G _max) calculated from the amiloride dose-response relationship. The ratio of G _amil in mucosal Na⁺ solutions to G _amil for mucosal K⁺ solutions was 22±6 for G _T measurements and 15±2 for G _max data. Serosal ion replacements in tissues treated with mucosal nystatin indicated a potassium conductance in the basolateral membrane. Equivalent circuit analyses of nystatin and amiloride data were used to resolve the cellular (E _c) and paracellular (R _j) resistances (5 kcm² and 8–9 kcm², respectively). Analysis I-V relationships for tissues depolarized with serosal K⁺ solutions revealed that the amiloride-sensitive pathway could be described as a Na⁺ conductance with a permeability coefficient (P _Na)=1.5±0.2× 10^–6 cm/s and the intracellular Na⁺ concentration (Na_i)=5±1 mM (n=5), similar to values from other tight epithelia. We conclude that A6 epithelia are capable of expressing large amiloride-sensitive currents which are highly Na⁺ selective.     

SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr346/356/366 phosphorylation

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr^346/356/366) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1–12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr^346/356/366 in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na⁺ conductance (G _Na) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr^346/356/366 phosphorylation. Although G _Na is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 μM) acutely activated SGK1 and the peak of this response (2–3 h) coincided with the induction of G _Na, and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G _Na, transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G _Na but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na⁺ absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G _Na, its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.     

Accelerated suicidal erythrocyte death in Klotho-deficient mice

Klotho, a membrane protein mainly expressed in parathyroid glands, kidney, and choroid plexus, counteracts aging and increases the life span. Accordingly, life span is significantly shorter in Klotho-deficient mice (klotho ^−/− ) than in their wild-type littermates (klotho ^+/+ ). The pleotropic effects of Klotho include inhibition of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) formation. Vitamin D-deficient diet reverses the shortening of life span in klotho ^−/− mice. In a variety of cells, 1,25(OH)₂D₃ stimulates Ca²⁺ entry. In erythrocytes, increased Ca²⁺ entry stimulates suicidal erythrocyte death, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. The present study explored the putative impact of Klotho on eryptosis. According to Fluo3 fluorescence, cytosolic Ca²⁺ concentration was significantly larger in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. According to annexin V-binding, phosphatidylserine exposure was significantly enhanced, and according to forward scatter, cell volume significantly decreased in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. Energy depletion (13 h glucose depletion) and oxidative stress (35 min 1 mM tert-butyl-hydroxyl-peroxide [tert-BOOH]) increased phosphatidylserine exposure to values again significantly larger in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. Reticulocyte number was significantly increased in klotho ^−/− mice, pointing to enhanced erythrocyte turnover. Vitamin D-deficient diet reversed the enhanced Ca²⁺ entry and annexin V-binding of klotho ^−/− erythrocytes. The present observations reveal a novel function of Klotho, i.e., the at least partially vitamin D-dependent regulation of cytosolic Ca²⁺ activity in and suicidal death of erythrocytes.     

Cooperative effect of ribosomal protein s19 and Pim-1 kinase on murine c-Myc expression and myeloid/erythroid cellularity

Diamond-Blackfan anemia is a bone marrow failure syndrome associated with heterozygous mutations in the ribosomal protein S19 (RPS19) gene in a subgroup of patients. One of the interacting partners with RPS19 is the oncoprotein PIM-1 kinase. We intercrossed Rps19 ^+/− and Pim-1 ^−/− mice strains to study the effect from the disruption of both genes. The double mutant (Rps19 ^+/− Pim-1 ^−/− ) mice display normal growth with increased peripheral white and red blood cell counts when compared to the w.t. mice (Rps19 ^+/+ Pim-1 ^+/+ ). Molecular analysis of bone marrow cells in Rps19 ^+/− Pim-1 ^−/− mice revealed up-regulated levels of c-Myc and the anti-apoptotic factors Bcl₂, Bcl_XL, and Mcl-1. This is associated with a reduction of the apoptotic factors Bak and Caspase 3 as well as the cell cycle regulator p21. Our findings suggest that combined Rps19 insufficiency and Pim-1 deficiency promote murine myeloid cell growth through a deregulation of c-Myc and a simultaneous up-regulation of anti-apoptotic Bcl proteins.     

Regulation of Na+/H+ exchanger in dendritic cells by Akt2

Dendritic cells (DCs) are antigen-presenting cells decisive in primary immune responses and establishment of immunological memory. They are activated by bacterial lipopolysaccharides (LPS), which lead to activation of Na⁺/H⁺ exchanger activity, cell swelling, reactive oxygen species (ROS) formation, and migration. The effects require functional phosphoinositide 3 kinase and are paralleled by Akt phosphorylation. The present study explored the putative involvement of the Akt isoform Akt2. To this end, experiments were performed in DCs isolated from bone marrow of mice lacking functional Akt2/PKB? (akt2 ^−/−) and respective wild-type animals (akt2 ^+/+). Based on BCECF fluorescence, cytosolic pH (pH_i) was significantly lower in akt2 ^−/− than in akt2 ^+/+ DCs. Transient exposure to NH₄Cl was followed by profound cytosolic acidification in both genotypes. Subsequent re-alkalinization was largely dependent on Na⁺ thus reflecting Na⁺/H⁺ exchanger activity and was significantly lower in akt2 ^−/− than in akt2 ^+/+ DCs. According to forward scatter in FACS analysis, cell volume was significantly lower in akt2 ^−/− than in akt2 ^+/+ DCs. Exposure of DCs to LPS led within 4 h to significant increases of Na⁺/H⁺ exchanger activity, cell volume, ROS production, and migration in akt2 ^+/+ mice, and its effects were significantly blunted in akt2 ^−/− DCs. The present observations disclose a role of Akt2 in the regulation of pH_i, cell volume, ROS production, and migration in dendritic cells.     

Active NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse does not require the presence of bicarbonate

The aim of the present study was to investigate whether bicarbonate buffer (CO₂ + HCO ₃ ^– ) is required to sustain maximal NaCl transport in the cortical thick ascending limb of Henle's loop (cTAL) of the mouse. Transepithelial Na⁺ and Cl^– net fluxes (J _Na, J _Cl, pmol min^–1 mm^–1), measured by electron microprobe analysis, were similar irrespective of the presence or absence of CO₂ + HCO ₃ ^– in luminal and bathing solutions J _NaCl with CO₂ + HCO ₃ ^– =203±25 pmol min^–1 mm^–1; J NaCl without CO₂ + HCO ₃ ^– =213±13 pmol min^–1 mm^–1, n=14). Furthermore the transepithelial potential difference, V _te, the transepithelial resistance, R _te, and the basolateral membrane potential, V _bl, were unaffected by CO₂ + HCO ₃ ^– . In the absence of CO₂ + HCO ₃ ^– , V _te was +17.0±1.7 mV(n=9) (lumen positive), R _te was 28±2 cm² (n=9) and V _bl was –76±4 mV (n=6). In the presence of CO₂ + HCO ₃ ^– , V _te, R _te and V _bl were +15.9±1.5 mV, 29±1 cm² and –73±5 mV, respectively. 4-Acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid (SITS; 0.1 mmol l^–1) and amiloride (1 mmol l^–1) added to the (CO₂ + HCO ₃ ^– )-containing lumen perfusate were without effect on V _te and R _te. Finally, the effect of furosemide (0.1 mmol l^–1) on V _te and V _bl in the presence of CO₂ + HCO ₃ ^– was investigated. Furosemide reversibly decreased V _te from +13.7±1.1 mV to +1.7±0.7 mV (n=6) and hyperpolarized V_bl from –70±1 to –89±3 mV (n=5), suggesting passive distribution of Cl^– across the basolateral membrane. In conclusion, these data suggest that active NaCl transport in the cTAL of the mouse does not require the presence of CO₂ + HCO ₃ ^– .     

PI3-Kinase-dependent electrogenic intestinal transport of glucose and amino acids

Intestinal glucose and amino acid transport is stimulated by the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B which are, in turn, stimulated following activation of the phosphoinositol-3 kinase (PI3 kinase). The present study has been performed to explore whether pharmacological inhibition of the PI3 kinase affects electrogenic jejunal transport of glucose and amino acids. In Ussing chamber experiments, glucose (20 mM), phenylalanine (20 mM), glutamine (20 mM), cysteine (20 mM), and proline (20 mM) generated lumen negative currents (I _glc, I _phe, I _gln, I _cys, and I _pro), respectively, which gradually declined following application of the PI3 kinase inhibitor Wortmannin (1 μM). Within 40 min, Wortmannin treatment significantly decreased I _glc by 39 ± 10% (n = 5), I _phe by 70 ± 7% (n = 4), I _gln by 69 ± 8% (n = 4), I _cys by 67 ± 8% (n = 6), and I _prol by 79 ± 12% (n = 3). A similar decline of I _glc was observed following application of the PI3 kinase inhibitor LY294002 (50 μM). Exposure to the inhibitors did not significantly alter transepithelial potential difference and resistance in the absence of substrates for electrogenic transport. The observations suggest that the electrogenic transport of glucose and several amino acids requires the continued activity of PI3 kinase. R. Rexhepaj and F. Artunc shared first authorship.