Triggering of suicidal erythrocyte death by uremic toxin indoxyl sulfate.

BACKGROUND: Anemia in end stage renal disease is attributed to impaired erythrocyte formation due to erythropoietin and iron deficiency. On the other hand, end stage renal disease enhances eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i) and by ceramide, which sensitizes erythrocytes to [Ca2+]i. Mechanisms triggering eryptosis in endstage renal disease remained enigmatic. The present study explored the effect of indoxyl sulfate, an uremic toxin accumulated in blood of patients with chronic kidney disease. METHODS: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, ceramide abundance by specific antibodies, hemolysis from hemoglobin release, and [Ca(2+)]i from Fluo3-fluorescence. RESULTS: A 48 hours exposure to indoxyl sulfate significantly increased [Ca(2+)]i (≥ 300 µM), significantly decreased forward scatter (≥ 300 µM) and significantly increased annexin-V-binding (≥ 50 µM). Indoxyl sulfate (150 µM) induced annexin-V-binding was virtually abolished in the nominal absence of extracellular Ca(2+). Indoxyl sulfate (150 µM) further enhanced ceramide abundance. CONCLUSION: Indoxyl sulfate stimulates suicidal erythrocyte death or eryptosis, an effect in large part due to stimulation of extracellular Ca(2+)entry with subsequent stimulation of cell shrinkage and cell membrane scrambling.

25-Hydroxyvitamin D3 1-α-hydroxylase-dependent stimulation of renal klotho expression by spironolactone.

BACKGROUND: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH)2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1) by FGF23. Conversely, 1,25(OH)2D3 stimulates, by activating the vitamin D3 receptor (Vdr), the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH)2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. METHODS: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293) or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA) and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR) or of mineralocorticoid receptor (NR3C2). RESULTS: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours) and mice (8 hours or 5 days). Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours). Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. CONCLUSION: Besides blocking the effects of aldosterone, spironolactone upregulates KLOTHO gene expression by upregulation of 25-hydroxyvitamin D3 1-alpha-hydroxylase with subsequent activation of the vitamin D3 receptor by 1,25(OH)2D3, an effect possibly independent from the mineralocorticoid receptor.

Sgk1-dependent stimulation of cardiac Na+/H+ exchanger Nhe1 by dexamethasone.

BACKGROUND/AIMS: The serum- and glucocorticoid-inducible kinase Sgk1 contributes to cardiac remodeling and development of heart failure, which is paralelled by Sgk1-dependent stimulation of the cardiac Na(+)/H(+) exchanger Nhe1. Glucocorticoids are powerful stimulators of Sgk1 expression and influence cardiac remodeling. The present study thus explored whether the glucocorticoid receptor agonist dexamethasone influenced cardiac Sgk1 expression, as well as activity, expression and phosphorylation at Ser(703) of the cardiac Na(+)/H(+) exchanger Nhe1. METHODS: Experiments were performed in HL-1 cardiomyocytes and gene targeted mice lacking functional Sgk1 (sgk1(-/-)) and respective wild type mice (sgk1(+/+)). Gene expression was determined by quantitative RT-PCR and Nhe1 phosphorylation was determined utilizing a specific antibody against a 14-3-3 binding motif at P-Ser(703), which represents a putative phosphorylation site recognition motif for Sgk1 and is involved in Nhe1 activation. Cytosolic pH (pHi) was determined utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Nhe activity by the Na(+)-dependent realkalinization after an ammonium pulse. RESULTS: Treatment of HL-1 cardiomyocytes with dexamethasone was followed by a significant increase in Sgk1 mRNA expression, parallelled by increased Na(+)/H(+) exchanger activity. Furthermore, dexamethasone significantly increased Nhe1 and Spp1 mRNA expression. The effects of dexamethasone were blunted by cotreatment of HL-1 cardiomyocytes with the Sgk1 inhibitor EMD638683. Cotreatment with Nhe1 inhibitor cariporide similarly prevented dexamethasone-stimulated Spp1 mRNA expression. In sgk1(+/+) mice, dexamethasone significantly increased cardiac Sgk1 mRNA levels. In sgk1(+/+) mice, but not in sgk1(-/-) mice, dexamethasone significantly increased cardiac Nhe1 mRNA expression and Nhe1 phosphorylation at Ser(703). Furthermore, cardiac Spp1, Ctgf, Nppa and Nppb mRNA levels were significantly increased in dexamethasone treated sgk1(+/+) mice, effects significantly blunted in sgk1(-/-) mice. CONCLUSIONS: Sgk1 is critically involved in the phosphorylation and activation of the cardiac Na(+)/H(+) exchanger Nhe1.

The uremic toxin acrolein promotes suicidal erythrocyte death.

BACKGROUND: Anemia is a major complication of end stage renal disease. The anemia is mainly the result of impaired formation of erythrocytes due to lack of erythropoietin and iron deficiency. Compelling evidence, however, points to the contribution of accelerated erythrocyte death, which decreases the life span of circulating erythrocytes. Erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). Erythrocytes could be sensitized to cytosolic Ca(2+) by ceramide. In end stage renal disease, eryptosis may possibly be stimulated by uremic toxins. The present study explored, whether the uremic toxin acrolein could trigger eryptosis. METHODS: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence, and ceramide from fluorescent antibodies. RESULTS: A 48 h exposure to acrolein (30 － 50 µM) did not significantly modify [Ca(2+)]i but significantly decreased forward scatter and increased annexin-V-binding. Acrolein further triggered slight, but significant hemolysis and increased ceramide formation in erythrocytes. Acrolein (50 µM) induced annexin-V-binding was significantly blunted in the nominal absence of extracellular Ca(2+). Acrolein augmented the annexin-V-binding following treatment with Ca(2+) ionophore ionomycin (1 µM). CONCLUSION: Acrolein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of ceramide formation with subsequent sensitisation of the erythrocytes to cytosolic Ca(2+).

Stimulation of suicidal erythrocyte death by increased extracellular phosphate concentrations.

BACKGROUND/AIM: Anemia in renal insufficiency results in part from impaired erythrocyte formation due to erythropoietin and iron deficiency. Beyond that, renal insufficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be stimulated by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). Several uremic toxins have previously been shown to stimulate eryptosis. Renal insufficiency is further paralleled by increase of plasma phosphate concentration. The present study thus explored the effect of phosphate on erythrocyte death. METHODS: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, and [Ca(2+)]i from Fluo3-fluorescence. RESULTS: Following a 48 hours incubation, the percentage of phosphatidylserine exposing erythrocytes markedly increased as a function of extracellular phosphate concentration (from 0-5 mM). The exposure to 2 mM or 5 mM phosphate was followed by slight but significant hemolysis. [Ca(2+)]i did not change significantly up to 2 mM phosphate but significantly decreased at 5 mM phosphate. The effect of 2 mM phosphate on phosphatidylserine exposure was significantly augmented by increase of extracellular Ca(2+) to 1.7 mM, and significantly blunted by nominal absence of extracellular Ca(2+), by additional presence of pyrophosphate as well as by presence of p38 inhibitor SB203580. CONCLUSION: Increasing phosphate concentration stimulates erythrocyte membrane scrambling, an effect depending on extracellular but not intracellular Ca(2+) concentration. It is hypothesized that suicidal erythrocyte death is triggered by complexed CaHPO4.

Sgk1 sensitivity of Na(+)/H(+) exchanger activity and cardiac remodeling following pressure overload.

Sustained increase of cardiac workload is known to trigger cardiac remodeling with eventual development of cardiac failure. Compelling evidence points to a critical role of enhanced cardiac Na(+)/H(+) exchanger (NHE1) activity in the underlying pathophysiology. The signaling triggering up-regulation of NHE1 remained, however, ill defined. The present study explored the involvement of the serum- and glucocorticoid-inducible kinase Sgk1 in cardiac remodeling due to transverse aortic constriction (TAC). To this end, experiments were performed in gene targeted mice lacking functional Sgk1 (sgk1 (-/-)) and their wild-type controls (sgk1 (+/+)). Transcript levels have been determined by RT-PCR, cytosolic pH (pH( i )) utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, Na(+)/H(+) exchanger activity by the Na(+)-dependent realkalinization after an ammonium pulse, ejection fraction (%) utilizing cardiac cine magnetic resonance imaging and cardiac glucose uptake by PET imaging. As a result, TAC increased the mRNA expression of Sgk1 in sgk1 (+/+) mice, paralleled by an increase in Nhe1 transcript levels as well as Na(+)/H(+) exchanger activity, all effects virtually abrogated in sgk1 (-/-) mice. In sgk1 (+/+) mice, TAC induced a decrease in Pgc1a mRNA expression, while Spp1 mRNA expression was increased, both effects diminished in the sgk1 (-/-) mice. TAC was followed by a significant increase of heart and lung weight in sgk1 (+/+) mice, an effect significantly blunted in sgk1 (-/-) mice. TAC increased the transcript levels of Anp and Bnp, effects again significantly blunted in sgk1 (-/-) mice. TAC increased transcript levels of Collagen I and III as well as Ctgf mRNA and CTGF protein abundance, effects significantly blunted in sgk1 (-/-) mice. TAC further decreased the ejection fraction in sgk1 (+/+) mice, an effect again attenuated in sgk1 (-/-) mice. Also, cardiac FDG-glucose uptake was increased to a larger extent in sgk1 (+/+) mice than in sgk1 (-/-) mice after TAC. These observations point to an important role for SGK1 in cardiac remodeling and development of heart failure following an excessive work load.

Enhanced catecholamine release in mice expressing PKB/SGK-resistant GSK3.

Glycogen synthase kinase 3 (GSK3) plays a decisive role in the regulation of multiple functions. GSK3 is phosphorylated and its activity inhibited by protein kinase B (PKB/Akt) and serum and glucocorticoid inducible kinase (SGK) isoforms, which are in turn activated by growth factors through phosphoinositide (PI) 3 kinase signaling. PI3/PKB/Akt/SGK-dependent inhibition of GSK3 is disrupted in gene-targeted knockin mice with mutated and thus PKB/SGK-resistant GSK3α,ß (gsk3 ( KI )) where the serine of the PKB/SGK phosphorylation site has been replaced by alanine. Recent experiments revealed that blood pressure is significantly higher in those mice than in wild type mice (gsk3 ( WT )). The present study was performed to elucidate the underlying cause. Blood pressure was determined with the tail cuff method, heart rate by ECG measurements, catecholamine concentrations by ELISA, and vanillylmandelic acid by high pressure liquid chromatography. As a result, blood pressure and heart rate were significantly higher in gsk3 ( KI ) than in gsk3 ( WT ) mice. The α-adrenergic blocker prazosin (1 µg/g body weight, b.w.) and the ganglion blocker hexamethonium (40 µg/g b.w.) decreased blood pressure to a larger extent in gsk3 ( KI ) than in gsk3 ( WT ) mice and virtually abrogated the difference between genotypes. Similarly, the ß-adrenergic blocker atenolol (5 µg/g b.w.) decreased the heart rate to a larger extent in gsk3 ( KI ) than in gsk3 ( WT ) mice and again dissipated the difference of heart rate between genotypes. Plasma epinephrine and norepinephrine concentrations, as well as urinary excretion of vanillylmandelic acid, were significantly higher in gsk3 ( KI ) than in gsk3 ( WT ) mice. The observations reveal a completely novel function of PKB/Akt/SGK-dependent GSK3 signaling, i.e., regulation of catecholamine release.