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Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of plateletderived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation., Introduction Untoward thrombus formation is associated with multiple major clinical disorders and is a leading cause of death and disability worldwide (1). Excessive platelet activation and aggregation at sites of [...]

Platelets are activated by increased cytsolic Ca2+ concentration ([Ca2+]i) following store-operated calcium entry (SOCE) accomplished by calcium release-activated calcium (CRAC) channel moiety Orail and its regulator STIM1. In other cells, Ca2+ transport is regulated by 1,25(OH) 2 vitamin D3 [1,25(OH) 2D3] 1,25(OH) 2D3 formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The prese study explored the effect of klotho deficiency o platelet Ca2+ signaling and activation. Platelets an megakaryocytes isolated from WT and kl/kl-mice we analyzed by RT-PCR, Western blotting, confocal microscopy, Fura-2-fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orail transcript and protein levels, SOCE, agonist-induce [Ca2+]i increase, activation-dependent degranulation, integrin αIIbβ3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27-90%). STIM1/Orail transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38-73%) 1,25(OH)2D3-treated WT megakaryocytes. Nuclear NF-ΚB subunit p50/p65 abundance was significant reduced in kl/kl-megakaryocytes (by 51-76%). Transfection with p50/p65 significantly increased STIM1 Orall transcript and protein levels in megakaryocytic MEG-01 cells (by 46-97%). Low-vitamin D diet (LVD of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocyte. Klotho deficiency inhibits platelet Ca2+ signaling and activation, an effect at least partially due to 1,25(OH)2D3-dependent down-regulation of NF-ΚB activity and STIM1/Orail expression in megakaryocytes. [ABSTRACT FROM AUTHOR]

Background/Aims: Renal tissue fibrosis contributes to the development of end-stage renal disease. Causes for renal tissue fibrosis include obstructive nephropathy. The development of renal fibrosis following unilateral ureteral obstruction (UUO) is blunted in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase SGK1. Similar to Akt isoforms, SGK1 phosphorylates and thus inactivates glycogen synthase kinase GSK-3. The present study explored whether PKB/SGK-dependent phoshorylation of GSK-3α/β impacts on pro-fibrotic signaling following UUO. Methods: UUO was induced in mice carrying a PKB/SGK-resistant GSK-3α/β (gsk-3KI) and corresponding wild-type mice (gsk-3WT). Three days after the obstructive injury, expression of fibrosis markers in kidney tissues was analyzed by quantitative RT-PCR and western blotting. Results: GSK-3α and GSK-3β phosphorylation was absent in both, the non-obstructed and the obstructed kidney tissues from gsk-3KI mice but was increased by UUO in kidney tissues from gsk-3WT mice. Expression of α-smooth muscle actin, type I collagen and type III collagen in the non-obstructed kidney tissues was not significantly different between gsk-3KI mice and gsk-3WT mice but was significantly less increased in the obstructed kidney tissues from gsk-3KI mice than from gsk-3WT mice. After UUO treatment, renal β-catenin protein abundance and renal expression of the β-catenin sensitive genes: c-Myc, Dkk1, Twist and Lef1 were again significantly less increased in kidney tissues from gsk-3KI mice than from gsk-3WT mice. Conclusions: PKB/SGK-dependent phosphorylation of glycogen synthase kinase GSK-3 contributes to the pro-fibrotic signaling leading to renal tissue fibrosis in obstructive nephropathy. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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 Ser703 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-Ser703, 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 Ser703. 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. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase (SPAK) regulates the renal thiazide sensitive NaCl cotransporter (NCC) and the renal furosemide sensitive Na+,K+,2Cl- cotransporter (NKCC2) and thus participates in the regulation of renal salt excretion, extracellular fluid volume and blood pressure. Inhibition of NCC leads to anticalciuria. Moreover, NCC is also expressed in osteoblasts where it is implicated in the regulation of bone mineralization. Osteoblasts further influence mineral metabolism by releasing the phosphaturic hormone FGF23. The present study explored, whether SPAK participates in the regulation of calcium-phosphate homeostasis. Methods: FGF23 serum levels and phosphate homeostasis were analyzed in gene targeted mice expressing SPAK resistant to WNK-dependent activation (spaktg/tg) and in mice expressing wild type SPAK (spakwt/wt). Results: Serum FGF23 level was significantly higher, urinary phosphate excretion significantly larger and serum phosphate concentration significantly lower in spaktg/tg mice than in spakwt/wt mice. Urinary calcium excretion was significantly decreased in spaktg/tg mice. Serum levels of calcitriol and PTH were not significantly different between the genotypes. Bone density was significantly increased in spaktg/tg mice compared to spakwt/wt mice. Treatment of spakwt/wt mice with HCT increased FGF23 serum levels, and led to phosphaturia and hypophosphatemia. Conclusions: SPAK is a strong regulator of FGF23 formation, bone mineralization and renal Ca2+ and phosphate excretion. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]