IntroductionPlatelet adhesion, activation, and aggregation are essential for primary hemostasis at sites of vascular injury but are also critically important for the development of acute thrombotic occlusion at regions of atherosclerotic plaque rupture, the major pathophysiologic mechanism underlying myocardial infarction and ischemic stroke. 1 Platelet activation is triggered by various agonists, including subendothelial collagen, ADP released from activated platelets, thrombin generated by the coagulation cascade, or the collagen receptor glycoprotein VI (GPVI)-specific agonists convulxin (CVX) and collagen-related peptide (CRP). 2 The agonists lead to platelet granule release, integrin ␣ IIb ␤ 3 activation, phosphatidylserine exposure, aggregation, and thrombus formation. 2 All those platelet responses depend on an increase of cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ), 3,4 which is accomplished by inositol-1,4,5-triphosphatemediated Ca 2ϩ release from intracellular stores triggering subsequent stimulation of store-operated Ca 2ϩ entry (SOCE) across the plasma membrane. 5 Two key players in platelet SOCE have recently been identified: The 4-transmembrane-spanning poreforming calcium release-activated channel moiety Orai1, which mediates entry of extracellular Ca 2ϩ , and stromal interaction molecule 1 (STIM1), an Orai1 regulating Ca 2ϩ sensor expressed predominantly in the endoplasmic reticulum. [6][7][8] Regulators of Orai1 in other cell types include receptor for activated protein kinase C-1, 9 reactive oxygen species, 10 and lipid rafts. 11 However, regulation of Orai1 in platelets is poorly understood. Platelet activation has been shown to be regulated in vitro and in vivo by the PI3K/Akt signaling cascade. 12,13 Interference with PI3K signaling has previously been shown to compromise Ca 2ϩ influx into platelets. 14,15 Signaling molecules regulated by PI3K signaling include the serum-and glucocorticoid-inducible kinase 1 (SGK1), a kinase belonging to the AGC family of serine/threonine protein kinases. 16,17 SGK1 has originally been cloned as a glucocorticoidsensitive gene but later shown to be regulated by a variety of hormones and other triggers, including thrombin, growth factors IGF-1 and TGF-␤, oxidative stress, and ischemia. 17 SGK1 has previously been reported to regulate a wide variety of carriers and ion channels, including the epithelial Ca 2ϩ channels TRPV5 and TRPV6. 17 Most recently, SGK1 has been shown to be critically important for the Ca 2ϩ entry into mast cells after activation of the IgE receptor, 18 an effect mediated by regulation of Orai1. 19 Furthermore, SGK1 participates in the regulation of renal tubular Na ϩ reabsorption, salt appetite, and thus blood pressure. 17 A gain-of-function SGK1 gene variant, the combined presence of single nucleotide polymorphism in intron 6 (rs1743966) and in exon 8 (rs1057293), is associated with enhanced blood pressure. 20 Submitted June 9, 2011; accepted August 28, 2011. Prepublished online as Blood First Edition paper, October 26, 2011; DOI 10.1182...

Klotho is a potent regulator of 1,25-hydroxyvitamin D3 [1,25(OH) 2 D 3 ] formation and calcium-phosphate metabolism. Klotho-hypomorphic mice (kl/kl mice) suffer from severe growth deficits, rapid aging, hyperphosphatemia, hyperaldosteronism, and extensive vascular and soft tissue calcification. Sequelae of klotho deficiency are similar to those of end-stage renal disease. We show here that the mineralocorticoid receptor antagonist spironolactone reduced vascular and soft tissue calcification and increased the life span of kl/kl mice, without significant effects on 1,25(OH) 2 D 3 , FGF23, calcium, and phosphate plasma concentrations. Spironolactone also reduced the expression of osteoinductive Pit1 and Tnfa mRNA, osteogenic transcription factors, and alkaline phosphatase (Alpl) in calcified tissues of kl/kl mice. In human aortic smooth muscle cells (HAoSMCs), aldosterone dose-dependently increased PIT1 mRNA expression, an effect paralleled by increased expression of osteogenic transcription factors and enhanced ALP activity. The effects of aldosterone were reversed by both spironolactone treatment and PIT1 silencing and were mitigated by FGF23 cotreatment in HAoSMCs. In conclusion, aldosterone contributes to vascular and soft tissue calcification, an effect due, at least in part, to stimulation of spironolactone-sensitive, PIT1-dependent osteoinductive signaling.

Klotho is a membrane protein participating in the inhibitory effect of FGF23 on the formation of 1,25-dihydroxyvitamin-D(3) [1,25(OH)(2)D(3)]. It participates in the regulation of renal tubular phosphate reabsorption and stimulates renal tubular Ca(2+) reabsorption. Klotho hypomorphic mice (klotho(hm)) suffer from severe growth deficit, rapid aging, and early death, events largely reversed by a vitamin D-deficient diet. The present study explored the role of Klotho deficiency in mineral and electrolyte metabolism. To this end, klotho(hm) mice and wild-type mice (klotho(+/+)) were subjected to a normal (D(+)) or vitamin D-deficient (D(-)) diet or to a vitamin D-deficient diet for 4 wk and then to a normal diet (D(-/+)). At the age of 8 wk, body weight was significantly lower in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice, klotho(hm)D(-) mice, and klotho(hm)D(-/+) mice. Plasma concentrations of 1,25(OH)(2)D(3,) adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), and aldosterone were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. Plasma volume was significantly smaller in klotho(hm)D(-/+) mice, and plasma urea, Ca(2+), phosphate and Na(+), but not K(+) concentrations were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. The differences were partially abrogated by a vitamin D-deficient diet. Moreover, the hyperaldosteronism was partially reversed by Ca(2+)-deficient diet. Ussing chamber experiments revealed a marked increase in amiloride-sensitive current across the colonic epithelium, pointing to enhanced epithelial sodium channel (ENaC) activity. A salt-deficient diet tended to decrease and a salt-rich diet significantly increased the life span of klotho(hm)D(+) mice. In conclusion, the present observation disclose that the excessive formation of 1,25(OH)(2)D(3) in Klotho-deficient mice results in extracellular volume depletion, which significantly contributes to the shortening of life span.

Urea cycle defects and acute or chronic liver failure are linked to systemic hyperammonemia and often result in cerebral dysfunction and encephalopathy. Although an important role of the liver in ammonia metabolism is widely accepted, the role of ammonia metabolizing pathways in the liver for maintenance of whole-body ammonia homeostasis in vivo remains ill-defined. Here, we show by generation of liver-specific Gln synthetase (GS)-deficient mice that GS in the liver is critically involved in systemic ammonia homeostasis in vivo. Hepatic deletion of GS triggered systemic hyperammonemia, which was associated with cerebral oxidative stress as indicated by increased levels of oxidized RNA and enhanced protein Tyr nitration. Liver-specific GS-deficient mice showed increased locomotion, impaired fear memory, and a slightly reduced life span. In conclusion, the present observations highlight the importance of hepatic GS for maintenance of ammonia homeostasis and establish the liver-specific GS KO mouse as a model with which to study effects of chronic hyperammonemia.hepatic encephalopathy | metabolic zonation | oxidative stress | RNA oxidation | glutamine

Klotho, a cofactor in suppressing 1,25(OH) 2 D 3 formation, is a powerful regulator of mineral metabolism. Klotho-hypomorphic mice (kl/kl) exhibit excessive plasma 1,25(OH) 2 D 3 , Ca 2+ , and phosphate concentrations, severe tissue calcification, volume depletion with hyperaldosteronism, and early death. Calcification is paralleled by overexpression of osteoinductive transcription factor Runx2/Cbfa1, Alpl, and senescenceassociated molecules Tgfb1, Pai-1, p21, and Glb1. Here, we show that NH 4 Cl treatment in drinking water (0.28 M) prevented soft tissue and vascular calcification and increased the life span of kl/kl mice .12-fold in males and .4-fold in females without significantly affecting extracellular pH or plasma concentrations of 1,25(OH) 2 D 3 , Ca 2+ , and phosphate. NH 4 Cl treatment significantly decreased plasma aldosterone and antidiuretic hormone concentrations and reversed the increase of Runx2/Cbfa1, Alpl, Tgfb1, Pai-1, p21, and Glb1 expression in aorta of kl/kl mice. Similarly, in primary human aortic smooth muscle cells (HAoSMCs), NH 4 Cl treatment reduced phosphate-induced mRNA expression of RUNX2/CBFA1, ALPL, and senescence-associated molecules. In both kl/kl mice and phosphate-treated HAoSMCs, levels of osmosensitive transcription factor NFAT5 and NFAT5-downstream mediator SOX9 were higher than in controls and decreased after NH 4 Cl treatment. Overexpression of NFAT5 in HAoSMCs mimicked the effect of phosphate and abrogated the effect of NH 4 Cl on SOX9, RUNX2/CBFA1, and ALPL mRNA expression. TGFB1 treatment of HAoSMCs upregulated NFAT5 expression and prevented the decrease of phosphate-induced NFAT5 expression after NH 4 Cl treatment. In conclusion, NH 4 Cl treatment prevents tissue calcification, reduces vascular senescence, and extends survival of klotho-hypomorphic mice. The effects of NH 4 Cl on vascular osteoinduction involve decrease of TGFB1 and inhibition of NFAT5-dependent osteochondrogenic signaling.