Die Rolle von Serin-Threonin-Kinasen für epitheliale Transportvorgänge

The phosphoinositide dependent kinase PDK1 activates the SGK isoforms SGK1, SGK2 and SGK3 and protein kinase B isoforms which in turn are known to stimulate a variety of sodium coupled transporters, such as the renal and intestinal Na+-dependent glucose transporter SGLT1. SGK1 is known to be up-regulated by mineralocorticoids and to enhance ENaC activity in several expression systems. Moreover, the amiloride-sensitive transepithelial potential difference in collecting duct is lower in gene-targeted mice lacking SGK1 (sgk1-/-) than in their wild type littermates (sgk1+/+). Accordingly, the ability of sgk1-/- mice to decrease urinary sodium output during salt depletion is impaired. ENaC activity and thus transepithelial potential difference in the colon are similarly influenced by mineralocorticoids. The first aim of the present study was to explore the role of PDK1 in in electrogenic glucose and amino-acid transport in small intestine and proximal renal tubules As mice completely lacking functional PDK1 are not viable, mice expressing 10-25% of PDK1 (pdk1hm) were compared to their wild type littermates (pdk1wt). Body weight was significantly smaller in pdk1hm than in pdk1wt mice. Despite lower body weight of pdk1hm mice, food and water intake were similar in pdk1hm and pdk1wt mice. Ussing chamber experiments showed that electrogenic transport of glucose as well as phenylalanine, cysteine, glutamine, proline, leucine and tryptophan was significantly smaller in jejunum of pdk1hm mice compared to pdk1wt mice. Similarly, proximal tubular electrogenic glucose transport as well as phenylalanine, glutamine and proline transport in isolated perfused renal tubule segments was decreased. Intraperitoneal injection of 3 g/kg bw glucose resulted in a similar increase of plasma glucose concentration in pdk1hm and in pdk1wt mice but led to a higher increase of urinary glucose excretion in pdk1hm mice. The urinary excretion of proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate and tryptophan was significantly larger in pdk1hm than in pdk1wt mice. According to immunoblotting of brush border membrane proteins prepared from kidney, expression of the Na+-dependent neutral amino acid transporter B0AT1 (SLC6A19), the glutamate transporter EAAC1/EAAT3 (SLC1A1) and the transporter for cationic amino acids and cystine b0,+AT (SLC7A9) was decreased but the Na+/proline cotransporter SIT (SLC6A20) was increased in pdk1hm mice. In conclusion, reduction of functional PDK1 leads to impairment of intestinal absorption and renal reabsorption of amino acids of electrogenic intestinal glucose absorption and renal glucose reabsorption. The combined intestinal and renal loss of amino acids may contribute to the growth defect of PDK1 deficient mice. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine. The next step was to look at the transepithelial potential (Vte) and the apparent amiloride-sensitive equivalent short circuit current (Iamil) in colon from sgk1-/- and sgk1+/+ mice. Both Vte and Iamil were significantly (p<0.05) higher in untreated sgk1-/- than in untreated sgk1+/+ mice under control diet. A 7 day exposure to low salt diet increased Vte and Iamil in both genotypes but did not abrogate the differences of Vte and Iamil between sgk1-/- and sgk1+/+ mice. Plasma aldosterone levels were significantly higher in sgk1-/- than in sgk1+/+ mice both under control conditions and under low salt diet. Treatment with dexamethasone (10µg/g BW) or with DOCA (1.5mg per day) significantly increased Vte and Iamil in sgk1+/+ mice but not in sgk1-/- mice. Under treatment with dexamethasone or DOCA Vte and Iamil were similar in sgk1-/- and sgk1+/+ mice. In conclusion, lack of SGK1 does not disrupt colonic ENaC activity and its regulation by salt depletion. Finally the functional significance of SGK3-dependent regulation of intestinal transport were studied. .Xenopus oocyte coexpression experiments revealed the capacity of SGK3 to up-regulate a variety of transport systems including the sodium-dependent glucose transporter SGLT1. 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 (Ig) 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.Die phosphoinositid-abhängige Kinase 1 (PDK1) aktiviert die SGK-Isoformen SGK1, SGK2 und SGK3 sowie die Isoformen der Proteinkinase B, die eine Vielzahl von Natrium-gekoppelten Transportern stimulieren wie z.B. den renalen und intestinalen glucose-Transporter SGLT1. SGK1 wird durch Mineralokortikoide hochreguliert und stimuliert die Aktivität des epithelialen Natrium-Kanals ENaC in verschiedenen Expressionssystemen. In SGK1 defizienten Mäusen (sgk1-/-) ist die amilorid-hemmbare transepitheliale Potentialdifferenz niedriger als in Wildtyp-Mäusen, unter Niedrigsalz-Diät ist die Fähigkeit zur Na-Konservierung in den sgk1-/- Mäusen eingeschränkt. Die ENaC-Aktivität im Kolon ist ähnlich wie in der Niere mineralokortikoid-abhängig. Das Ziel der ersten Studie war es, den Einfluss der PDK1 am elektrogenen Glukose- und Aminosäurentransport im Dünndarm und im proximalen Tubulus zu untersuchen. Da Mäuse mit vollständigem PDK1-Verlust nicht lebensfähig sind, wurden PDK1-hypomorphe Mäuse (pdk1hm) mit einer PDK1-Restaktivität von 10-25% untersucht. Das Körpergewicht der PDK1-hypomorphen Mäuse war signifikant geringer als dasjenige der Wildtyp-Tiere, die Futter- und Flüssigkeitsaufnahme waren jedoch ähnlich hoch. Ussing-Kammer-Experimente zeigten einen reduzierten elektrogenen Transport für Glukose sowie für die Aminosäuren Phenylalanin, Cystein, Glutamin, Prolin, Leucin und Tryptophan in pdk1hm -Mäusen verglichen mit pdk1wt Mäusen. Analog dazu war der elektrogene Transport im isolierten proximalen Tubulus für Glucose sowie für Phenylalanin, Glutamin und Prolin vermindert. Unter intraperitonealer Beladung mit 3 g/kg Glucose kam es zu einer Glukosurie in pdk1hm -Mäusen, trotz ähnlich hoher Glukose-Spiegel wie in Wildtyp-Mäusen. Die Urinausscheidung von Prolin, Valin, Guanidinoacetat, Methionin, Phenylalanin, Citrullin, Glutamine/Glutamat und Tryptophan war in pdk1hm -Mäusen signifikant höher als in Widltyp-Mäusen. Im Western Blot von renalen Bürstensaum-Membranen von pdk1hm Mäusen war die Expression der Na+-abhängigen neutralen Aminosäuren-transporter B0AT1 (SLC6A19), des Glutamat-Transporters EAAC1/EAAT3 (SLC1A1) sowie des Transporter für kationische Aminosäuren und Cystin b0,+AT (SLC7A9) erniedrigt, die Expression des Na+-Prolin Cotransporters SIT (SLC6A20) erhöht. Zusammenfassend konnte gezeigt werden, dass eine Reduktion der PDK1 zu einer verminderten intestinalen Absorption sowie renalen Reabsorption von Glukose und Aminosäuren führte, was auf einen bisher nicht bekannten Regulationsweg hinweist. Der kombinierte intestinale und renale Verlust von Aminosäuren und Glukose könnte zum Minderwuchs der PDK1 hypomorphen Mäuse beitragen. In weiteren Untersuchungen wurde die transepitheliale Potentialdifferenz (Vte) und der amilorid-hemmbare Kurzschluss-Strom (Iamil) im Kolon von SGK1 defizienten (sgk1-/-) und Wildtyp-Mäusen untersucht. Sowohl Vte und Iamil waren in unbehandelten sgk1-/--Mäusen unter Kontrolldiät signifikant höher als in sgk1+/+ Mäusen. Eine 7-tägige Behandlung mit einer Niedrigsalz-Diät erhöhte Vte und Iamil in beiden Genotypen, konnte den Unterschied in Vte und Iamil zwischen sgk1-/-- und Wildtyp-Mäusen jedoch nicht aufheben. Plasma-Aldosteron-Spiegel waren in sgk1-/- -Mäusen sowohl unter Kontroll- wie Niedrigsalz-Diät signifikant höher. Behandlung mit Dexamethason (10µg/g) oder mit DOCA (1.5mg/ die) erhöhte Vte und Iamil nur in Wildtyp-Mäusen, jedoch nicht in sgk1-/- Mäusen. Unter Behandlung mit Dexamethason oder DOCA waren sowohl Vte als auch Iamil in sgk1-/- und Wildtyp-Mäusen ähnlich hoch. Die Ergebnisse zeigen zusammengenommen, dass ein Fehlen der SGK1 nicht die Aktivität und Regulation des ENaCs im Kolon unterbricht. Zuletzt wurde die funktionelle Bedeutung der SGK3 in der Regulation des intestinalen Transports untersucht. Zuvor hatten Experimente im Xenopus-Expressionssystem gezeigt, dass die SGK3 eine Vielzahl von Transportern beeinflussen kann, u.a. den Glucosetransporters SGLT1. In SGK3-defizienten Mäusen (sgk3-/-) zeigte sich verglichen mit Wildtyp-Mäusen eine höhere Futteraufnahme und ein höheres Stuhltrockengewicht. Die Glukose-induzierten Ströme (Iglc) waren im Jejunum signifikant geringer in sgk3-/- - Mäusen als in Wildtyp-Tieren. Der Nüchtern-Blutzucker war in sgk3-/- -Mäusen signifikant niedriger. Der intestinale elektrogene Transport von Phenylalanin, Cystein, Glutamin und Prolin ware hingegen zwischen sgk3-/- und Wildtyp-Mäusen nicht verschieden. Daher kann gefolgert warden, dass die SGK3 für die intestinale Na+-gekoppelte Glukoseaufnahme erforderlich ist und dass eine verminderte Glukoseaufnahme für die Wachstumsretardierung und die niedrigen Blutzuckerwerte verantwortlich sein könnte, was zu einer kompensatorischen Zunahme der Futteraufnahme führen könnte

Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2

Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. Spontaneous PS exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death

Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2

Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. Spontaneous PS exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death.Fil: Bissinger, Rosi. Eberhard Karls Universität Tübingen; AlemaniaFil: Lang, Elisabeth. Universitat Dusseldorf; AlemaniaFil: Ghashghaeinia, Mehrdad. Eberhard Karls Universität Tübingen; AlemaniaFil: Singh, Yogesh. Eberhard Karls Universität Tübingen; AlemaniaFil: Zelenak, Christine. Charité Medical University; AlemaniaFil: Fehrenbacher, Birgit. Eberhard Karls Universität Tübingen; AlemaniaFil: Honisch, Sabina. Eberhard Karls Universität Tübingen; AlemaniaFil: Chen, Hong. Eberhard Karls Universität Tübingen; AlemaniaFil: Fakhri, Hajar. Eberhard Karls Universität Tübingen; AlemaniaFil: Umbach, Anja T.. Eberhard Karls Universität Tübingen; AlemaniaFil: Liu, Guilai. Eberhard Karls Universität Tübingen; AlemaniaFil: Rexhepaj, Rexhep. Universitat Bonn; AlemaniaFil: Liu, Guoxing. Eberhard Karls Universität Tübingen; AlemaniaFil: Schaller, Martin. Eberhard Karls Universität Tübingen; AlemaniaFil: Mack, Andreas F.. Eberhard Karls Universität Tübingen; AlemaniaFil: Lupescu, Adrian. Eberhard Karls Universität Tübingen; AlemaniaFil: Birnbaumer, Lutz. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Lang, Florian. Eberhard Karls Universität Tübingen; AlemaniaFil: Qadri, Syed M.. Eberhard Karls Universität Tübingen; Alemania. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentin

Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion.

To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2

Vitamin D-Rich Diet in Mice Modulates Erythrocyte Survival

Background/Aims: Epidemiological evidence suggests that vitamin D deficiency is associated with anemia. The potent metabolite 1,25(OH)2 vitamin D3 [1,25(OH)2D3] activates various signaling cascades regulating a myriad of cellular functions including suicidal cell death or apoptosis. Suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Stimulation of eryptosis may limit lifespan of circulating erythrocytes and thus cause anemia. In the present study, we explored the effect of a high vitamin D diet (10,000 I.U. vitamin D for 14 days) in mice on eryptosis. Methods: Plasma concentrations of erythropoietin were estimated using an immunoassay kit, blood count using an electronic hematology particle counter, relative reticulocyte numbers using Retic-COUNT® reagent, PS exposure at the cell surface from annexin V binding, cell volume from forward scatter, and cytosolic Ca2+ ([Ca2+]i) from Fluo3-fluorescence in FACS analysis. Results: Vitamin D treatment decreased mean corpuscular volume, reticulocyte count, and plasma erythropoietin levels. Vitamin D treatment slightly but significantly decreased forward scatter but did not significantly modify spontaneous PS exposure and [Ca2+]i of freshly drawn erythrocytes. Vitamin D treatment augmented the stimulation of PS exposure and cell shrinkage following exposure to hyperosmotic shock (addition of 550 mM sucrose) or energy depletion (glucose removal) without significantly modifying [Ca2+]i. Conclusions: The present observations point to a subtle effect of exogenous vitamin D supplementation on erythrocyte survival

Responses to Diuretic Treatment in Gene-Targeted Mice Lacking Serum- and Glucocorticoid-Inducible Kinase 1

Background/Aims: Serum- and glucocorticoid-inducible kinase 1 (SGK1) stimulates the epithelial sodium channel (ENaC), renal outer medullary K + channel 1, Na + /K + -ATPase and presumably the Na + -Cl – cotransporter (NCC). SGK1-deficient mice (s gk– /– ) show a compensated salt-losing phenotype with secondary hyperaldosteronism. The present experiments explored the role of SGK1 in the response to diuretics. Methods: sgk1 –/– mice and their wild-type littermates (s gk1+ /+ ) were treated with the ENaC blocker triamterene (200 mg/l), the Na + -K + -2Cl – cotransport inhibitor furosemide (125 mg/l), the NCC blocker hydrochlorothiazide (400 mg/l) and the mineralocorticoid receptor blocker canrenoate (800 mg/l) for 8 days. Renal SGK1 expression was studied using quantitative RT-PCR and immunofluorescence. Results: Diuretic treatment increased SGK1 mRNA and protein expression in the kidney of wild-type sgk1+ /+ mice. The responses to furosemide, hydrochlorothiazide or canrenoate were not different between s gk1+ /+ and sgk1 –/– mice, and were accompanied by moderate increases in plasma aldosterone and urea concentrations. However, treatment with triamterene in sgk1 –/– mice (but not in sgk1 +/+ mice) led to severe, eventually lethal, body weight loss as well as increases in plasma aldosterone, urea and K + concentrations. Conclusions: SGK1 is required for diuretic tolerance to triamterene. The observations confirm the impaired kaliuretic potency of sgk1 –/– mice and point to a role of SGK1 in renal Na + reabsorption by mechanisms other than ENaC