Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Mice.

Chronic kidney disease (CKD) is associated with various pathologic changes, including elevations in serum phosphate levels (hyperphosphatemia), vascular calcification, and skeletal muscle atrophy. Elevated phosphate can damage vascular smooth muscle cells and cause vascular calcification. Here, we determined whether high phosphate can also affect skeletal muscle cells and whether hyperphosphatemia, in the context of CKD or by itself, is associated with skeletal muscle atrophy. As models of hyperphosphatemia with CKD, we studied mice receiving an adenine-rich diet for 14 weeks and mice with deletion of Collagen 4a3 (Col4a3-/-). As models of hyperphosphatemia without CKD, we analyzed mice receiving a high-phosphate diet for three and six months as well as a genetic model for klotho deficiency (kl/kl). We found that adenine, Col4a3-/-, and kl/kl mice have reduced skeletal muscle mass and function and develop atrophy. Mice on a high-phosphate diet for six months also had lower skeletal muscle mass and function but no significant signs of atrophy, indicating less severe damage compared with the other three models. To determine the potential direct actions of phosphate on skeletal muscle, we cultured primary mouse myotubes in high phosphate concentrations, and we detected the induction of atrophy. We conclude that in experimental mouse models, hyperphosphatemia is sufficient to induce skeletal muscle atrophy and that, among various other factors, elevated phosphate levels might contribute to skeletal muscle injury in CKD.

Acute cardiac overload does not induce cardiac or skeletal expression of fibroblast growth factor 23 in rats.

Elevated fibroblast growth factor 23 (FGF23) is associated with cardiovascular events, particularly heart failure. Although FGF23 has been reported to induce cardiac hypertrophy, recent studies demonstrated that cardiac hypertrophy and myocardial infarction induce FGF23 production by cardiomyocytes. We aimed to explore whether acute cardiac overload increases cardiac and skeletal FGF23 expression and circulating FGF23 levels. METHODS: We administered 30 µL/g bodyweight of isotonic saline intraperitoneally in rats to induce acute cardiac overload. We measured serum FGF23 levels and other parameters of mineral metabolism at 2, 6, and 24 h after saline or sham injection. We also analyzed gene expression in the heart, calvarium, femur, and kidney at 2 and 24 h after injection. RESULTS: Acute saline injection induced cardiac overload as evidenced by a significant upregulation of brain natriuretic peptide along with a trend towards increased expression of atrial natriuretic peptide and mild hyponatremia. However, there were no changes in serum FGF23 levels or FGF23 expression in the heart, calvarium, or femur. CONCLUSIONS: Acute cardiac overload by saline injection in rats did neither induce FGF23 expression in the heart or bone nor did it increase serum FGF23 levels. These findings suggest that more severe or long-term cardiac damage is required for induction of FGF23 expression.

Fetuin-A, fibroblast growth factor 23 and inflammation in critically ill patients with sepsis.

Fetuin-A is a glycoprotein with multifaceted roles, produced mainly in the liver. FGF23 has been reported to control Fetuin-A production in hepatocytes and in the bone. Furthermore, several studies have showed that higher circulating FGF23 levels stimulate inflammatory cytokines in the liver. However, the mechanistic insights linking bilirubin-Fetuin-A, FGF23 and inflammation in patients with sepsis is poorly understood. Therefore, further experimental research is required to link Fetuin, FGF23 and inflammation in the animal models of sepsis to gain further mechanistic insight.

Anticonvulsive effects of protodioscin against pilocarpine-induced epilepsy.

Epilepsy is associated with increased morbidity and mortality together and places a large financial burden on individuals and society. To evaluate the anticonvulsant action of protodioscin (PDSN) in experiments with animals with pilocarpine-induced convulsions. We assessed the activity of PDSN in pilocarpine induced seizures in combination with different agents which are acting via diverse receptors, such as atropine, memantine, nimodipine, diazepam, and flumazenil, to determine the exact receptors responsible for the action of PDSN. Furthermore, the level of antioxidant markers was investigated in the cerebellum and cerebral cortex in mice to define the antioxidant action of PDSN. The effects of PDSN on proapoptotic markers (i.e., Bcl-2, Bax, and caspase-3) was investigated via western blot analysis. PDSN significantly enhanced latency to the first convulsion and survival compared to the group treated with pilocarpine alone. Moreover, PDSN improved animal survival, and subjects experiencing no convulsions. Striatal glutamate and aspartate levels were not modified, and gamma amino butyric acid (GABA) levels increased, as a result of treatment with PDSN. The results suggest that the anticonvulsive action of PDSN is dependent on inhibitory amino acids. PDSN treatment also significantly decreased nitrite levels in the blood and brain cortex compared to the normal control. In the western blot analysis, PDSN exerted its neuroprotective effect via the upregulation of Bcl-2 and downregulation of Bax and caspase-3. The results of this study suggest that PDSN exerts neuroprotective effects via multiple mechanisms.

Insulin suppresses the production of fibroblast growth factor 23 (FGF23).

Fibroblast growth factor 23 (FGF23) is produced by bone cells and regulates renal phosphate and vitamin D metabolism, as well as causing left ventricular hypertrophy. FGF23 deficiency results in rapid aging, whereas high plasma FGF23 levels are found in several disorders, including kidney or cardiovascular diseases. Regulators of FGF23 production include parathyroid hormone (PTH), calcitriol, dietary phosphate, and inflammation. We report that insulin and insulin-like growth factor 1 (IGF1) are negative regulators of FGF23 production. In UMR106 osteoblast-like cells, insulin and IGF1 down-regulated FGF23 production by inhibiting the transcription factor forkhead box protein O1 (FOXO1) through phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB)/Akt signaling. Insulin deficiency caused a surge in the serum FGF23 concentration in mice, which was reversed by administration of insulin. In women, a highly significant negative correlation between FGF23 plasma concentration and increase in plasma insulin level following an oral glucose load was found. Our results provide strong evidence that insulin/IGF1-dependent PI3K/PKB/Akt/FOXO1 signaling is a powerful suppressor of FGF23 production in vitro as well as in mice and in humans.

A high-fat diet stimulates fibroblast growth factor 23 formation in mice through TNFα upregulation.

BACKGROUND/OBJECTIVES: Bone-derived fibroblast growth factor 23 (FGF23) is a hormone that suppresses renal phosphate reabsorption and calcitriol (i.e., 1,25(OH)2D3) formation together with its co-receptor Klotho. FGF23- or Klotho-deficient mice suffer from rapid aging with multiple age-associated diseases, at least in part due to massive calcification. FGF23 is considered as a disease biomarker since elevated plasma levels are observed early in patients with acute and chronic disorders including renal, cardiovascular, inflammatory, and metabolic diseases. An energy-dense diet, which induces sequelae of the metabolic syndrome in humans and mice at least in part by enhancing pro-inflammatory TNFα formation, has recently been demonstrated to stimulate FGF23 production. METHODS: We investigated the relevance of TNFα for high-fat diet (HFD)-induced FGF23 formation in wild-type (tnf+/+) and TNFα-deficient (tnf-/-) mice. RESULTS: Within 3 weeks, HFD feeding resulted in a strong increase in the serum FGF23 level in tnf+/+ mice. Moreover, it caused low-grade inflammation as evident from a surge in hepatic Tnfα transcript levels. TNFα stimulated Fgf23 transcription in UMR106 osteoblast-like cells. Serum FGF23 was significantly lower in tnf-/- mice compared to tnf+/+ mice following HFD. Serum phosphate and calcitriol were not significantly affected by genotype or diet. CONCLUSIONS: We show that HFD feeding is a powerful stimulator of murine FGF23 production through TNFα formation.

miR-204 suppresses the development and progression of human glioblastoma by targeting ATF2.

In human cancers, miRNAs are important regulators of multiple cellular processes, and aberrant miRNA expression has been observed, and their alterations contribute to multiple cancer development and progression. Till now, little has been known about the role of miR-204 in human glioblastoma (GBM). In the present study, we used in-vitro assays to investigate the mechanisms of miR-204 in GBM cell lines and 60 cases of GBM tissues. Here, we found that miR-204 expression is downregulated in both GBM cell lines A172, U87 and U251 cells and GBM tissues as compared with NHA cells and normal tissues (all p<0.001). In addition, the ectopic expression of miR-204 suppressed A172 and U87 cell proliferation, migration and invasion. Meanwhile, miR-204 over-expression extremely inhibited the protein expression of ATF2. Notably, the enforced expression of ATF2 in A172 and U87 cells with the over-expression of miR-204 attenuated the inhibitory effects of miR-204 on proliferation, migration and invasion. In conclusion, our findings suggest that miR-204 suppressed cell proliferation, migration and invasion through inhibition of ATF2, thus, miR-204 may function as a useful drug target in the treatment and diagnosis of GBM.

PI3K-resistant GSK3 controls adiponectin formation and protects from metabolic syndrome.

Metabolic syndrome is characterized by insulin resistance, obesity, and dyslipidemia. It is the consequence of an imbalance between caloric intake and energy consumption. Adiponectin protects against metabolic syndrome. Insulin-induced signaling includes activation of PI3 kinase and protein kinase B (PKB)/Akt. PKB/Akt in turn inactivates glycogen synthase kinase (GSK) 3, a major regulator of metabolism. Here, we studied the significance of PI3K-dependent GSK3 inactivation for adiponectin formation in diet-induced metabolic syndrome. Mice expressing PI3K-insensitive GSK3 (gsk3(KI)) and wild-type mice (gsk3(WT)) were fed a high-fat diet. Compared with gsk3(WT) mice, gsk3(KI) mice were protected against the development of metabolic syndrome as evident from a markedly lower weight gain, lower total body and liver fat accumulation, better glucose tolerance, stronger hepatic insulin-dependent PKB/Akt phosphorylation, lower serum insulin, cholesterol, and triglyceride levels, as well as higher energy expenditure. Serum adiponectin concentration and the activity of transcription factor C/EBPα controlling the expression of adiponectin in adipose tissue was significantly higher in gsk3(KI) mice than in gsk3(WT) mice. Treatment with GSK3 inhibitor lithium significantly decreased the serum adiponectin concentration of gsk3(KI) mice and abrogated the difference in C/EBPα activity between the genotypes. Taken together, our data demonstrate that the expression of PI3K-insensitive GSK3 stimulates the production of adiponectin and protects from diet-induced metabolic syndrome.

Fibroblast growth factor (Fgf) 23 gene transcription depends on actin cytoskeleton reorganization.

FGF23 regulates renal phosphate and vitamin D metabolism. Loss of FGF23 results in massive calcification and rapid aging. FGF23 production is stimulated by 1,25(OH)2D3 and NFκB signaling. Here, we report that treatment of UMR106 osteoblast-like cells with 1,25(OH)2D3, inducing Fgf23 transcription, resulted in actin polymerization which was blocked by NFκB inhibitor wogonin. Interestingly, 1,25(OH)2D3-induced Fgf23 gene transcription was abolished by the actin microfilament-disrupting agent cytochalasin B, as well as by the inhibition of actin-regulating Rac1/PAK1 signaling. Our results provide strong evidence that actin redistribution regulated by the Rac1/PAK1 pathway participates in 1,25(OH)2D3-induced Fgf23 gene transcription.

Up-regulation of FGF23 release by aldosterone.

The fibroblast growth factor (FGF23) plasma level is high in cardiac and renal failure and is associated with poor clinical prognosis of these disorders. Both diseases are paralleled by hyperaldosteronism. Excessive FGF23 levels and hyperaldosteronism are further observed in Klotho-deficient mice. The present study explored a putative aldosterone sensitivity of Fgf23 transcription and secretion the putative involvement of the aldosterone sensitive serum & glucocorticoid inducible kinase SGK1, SGK1 sensitive transcription factor NFκB and store operated Ca(2+) entry (SOCE). Serum FGF23 levels were determined by ELISA in mice following sham treatment or exposure to deoxycorticosterone acetate (DOCA) or salt depletion. In osteoblastic UMR106 cells transcript levels were quantified by qRT-PCR, cytosolic Ca(2+) concentration utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, DOCA treatment and salt depletion of mice elevated the serum C-terminal FGF23 concentration. In UMR106 cells aldosterone enhanced and spironolactone decreased SOCE. Aldosterone further increased Fgf23 transcript levels in UMR106 cells, an effect reversed by mineralocorticoid receptor blockers spironolactone and eplerenone, SGK1 inhibitor EMD638683, NFκB-inhibitor withaferin A, and Ca(2+) channel blocker YM58483. In conclusion, Fgf23 expression is up-regulated by aldosterone, an effect sensitive to SGK1, NFκB and store-operated Ca(2+) entry.

NFκB-sensitive Orai1 expression in the regulation of FGF23 release.

UNLABELLED: Fibroblast growth factor (FGF23) plasma levels are elevated in cardiac and renal failure and correlate with poor clinical prognosis of those disorders. Both disorders are associated with inflammation and activation of the inflammatory transcription factor NFκB. An excessive FGF23 level is further observed in Klotho-deficient mice. The present study explored a putative sensitivity of FGF23 expression to transcription factor NFκB, which is known to upregulate Orai1, the Ca(2+) channel accomplishing store-operated Ca(2+) entry (SOCE). In osteoblastic cells (UMR106) and immortalized primary periosteal (IPO) cells, protein abundance was determined by Western blotting, and in UMR106 cells, transcript levels were quantified by RT-PCR, cytosolic Ca(2+) activity utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, UMR106 and IPO cells expressed Ca(2+) channel Orai1. SOCE was lowered by NFκB inhibitor wogonin as well as by Orai1 inhibitors 2-APB and YM58483. UMR106 cell Fgf23 transcripts were increased by stimulation of SOCE and Ca(2+) ionophore ionomycin and decreased by Orai inhibitors 2-APB, YM58483 and SK&F96365, by Orai1 silencing, as well as by NFκB inhibitors wogonin, withaferin A, and CAS 545380-34-5. In conclusion, Fgf23 expression is upregulated by stimulation of NFκB-sensitive, store-operated Ca(2+) entry. KEY MESSAGES: Osteoblast UMR106 and IPO cells express Ca(2+) channel Orai1. Osteoblast store-operated Ca(2+) entry is accomplished by NFκB-sensitive Orai1. Osteoblast Fgf23 transcription is upregulated by increase in the cytosolic Ca(2+) activity. Fgf23 transcription is decreased by Orai inhibitors and Orai1 silencing. Fgf23 transcription is lowered by NFκB inhibitors.

Enhanced FGF23 production in mice expressing PI3K-insensitive GSK3 is normalized by ß-blocker treatment.

Glycogen synthase kinase (GSK)-3 is a ubiquitously expressed kinase inhibited by insulin-dependent Akt/PKB/SGK. Mice expressing Akt/PKB/SGK-resistant GSK3α/GSK3ß (gsk3(KI)) exhibit enhanced sympathetic nervous activity and phosphaturia with decreased bone density. Hormones participating in phosphate homeostasis include fibroblast growth factor (FGF)-23, a bone-derived hormone that inhibits 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; calcitriol) formation and phosphate reabsorption in the kidney and counteracts vascular calcification and aging. FGF23 secretion is stimulated by the sympathetic nervous system. We studied the role of GSK3-controlled sympathetic activity in FGF23 production and phosphate metabolism. Serum FGF23, 1,25(OH)2D3, and urinary vanillylmandelic acid (VMA) were measured by ELISA, and serum and urinary phosphate and calcium were measured by photometry in gsk3(KI) and gsk3(WT) mice, before and after 1 wk of oral treatment with the ß-blocker propranolol. Urinary VMA excretion, serum FGF23, and renal phosphate and calcium excretion were significantly higher, and serum 1,25(OH)2D3 and phosphate concentrations were lower in gsk3(KI) mice than in gsk3(WT) mice. Propranolol treatment decreased serum FGF23 and loss of renal calcium and phosphate and increased serum phosphate concentration in gsk3(KI) mice. We conclude that Akt/PKB/SGK-sensitive GSK3 inhibition participates in the regulation of FGF23 release, 1,25(OH)2D3 formation, and thus mineral metabolism, by controlling the activity of the sympathetic nervous system.

Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2.

The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk(-/-)) and corresponding wild-type mice (msk(+/+)). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk(-/-) and msk(+/+) mice, but reticulocyte count was significantly increased in msk(-/-) mice. Cell membrane PS exposure was similar in untreated msk(-/-) and msk(+/+) erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk(-/-) erythrocytes than in msk(+/+) erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk(-/-) erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk(-/-) mice. The spleens from msk(-/-) mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk(+/+) mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice.

Janus kinase 3 regulates renal 25-hydroxyvitamin D 1α-hydroxylase expression, calcitriol formation, and phosphate metabolism.

Calcitriol, a powerful regulator of phosphate metabolism and immune response, is generated by 25-hydroxyvitamin D 1α-hydroxylase in the kidney and macrophages. Renal 1α-hydroxylase expression is suppressed by Klotho and FGF23, the expression of which is stimulated by calcitriol. Interferon γ (INFγ) regulates 1α-hydroxylase expression in macrophages through transcription factor interferon regulatory factor-1. INFγ-signaling includes Janus kinase 3 (JAK3) but a role of JAK3 in the regulation of 1α-hydroxylase expression and mineral metabolism has not been shown. Thus, the impact of JAK3 deficiency on calcitriol formation and phosphate metabolism was measured. Renal interferon regulatory factor-1 and 1α-hydroxylase transcript levels, serum calcitriol and FGF23 levels, intestinal phosphate absorption as well as absolute and fractional renal phosphate excretion were significantly higher in jak3 knockout than in wild-type mice. Coexpression of JAK3 increased the phosphate-induced current in renal sodium-phosphate cotransporter-expressing Xenopus oocytes. Thus, JAK3 is a powerful regulator of 1α-hydroxylase expression and phosphate transport. Its deficiency leads to marked derangement of phosphate metabolism.

Influence of annexin A7 on insulin sensitivity of cellular glucose uptake.

Insulin sensitivity is decreased by prostaglandin E2 (PGE2), a major product of cyclooxygenase (COX). As shown in erythrocytes, PGE2 formation is inhibited by annexin A7. The present study defined the role of annexin A7 in glucose metabolism. Gene-targeted mice lacking annexin A7 (annexin7 (-/-)) were compared to wild-type mice (annexin7 (+/+)). The serum 6-Keto-prostaglandin-F1α (6-Keto-PGF1α) concentration was measured by ELISA and hepatic COX activity determined by an enzyme assay. Expression of COX-1, COX-2, prostaglandin E synthase, GLUT-4, and insulin receptor was determined by Western blotting. Glucose and insulin serum concentrations were analyzed following an intraperitoneal glucose load and glucose serum levels after intraperitoneal injection of insulin. Experiments were done without and with pretreatment of the mice with COX-inhibitor aspirin. The serum 6-Keto-PGF1α level and hepatic COX activity were significantly higher in annexin7 (-/-) than in annexin7 (+/+) mice. Hepatic COX-1 expression was higher in annexin7 (-/-) mice. Glucose tolerance was decreased in annexin7 (-/-) mice. Intraperitoneal insulin injection decreased the serum glucose level in both genotypes, an effect significantly less pronounced in annexin7 (-/-) mice. Glucose-induced insulin secretion was higher in annexin7 (-/-) mice. GLUT-4 expression in skeletal muscle from annexin7 (-/-) mice was reduced. Aspirin pretreatment lowered the increase in insulin concentration following glucose injection in both genotypes and virtually abrogated the differences in serum insulin between the genotypes. Aspirin pretreatment improved glucose tolerance in annexin7 (-/-) mice. In conclusion, annexin A7 influences insulin sensitivity of cellular glucose uptake and thus glucose tolerance. These effects depend on COX activity.

Checkpoint kinase Chk2 controls renal Cyp27b1 expression, calcitriol formation, and calcium-phosphate metabolism.

Checkpoint kinase 2 (Chk2) is the main effector kinase of ataxia telangiectasia mutated (ATM) and responsible for cell cycle regulation. ATM signaling has been shown to upregulate interferon-regulating factor-1 (IRF-1), a transcription factor also expressed in the kidney. Calcitriol (1,25 (OH)2D3), a major regulator of mineral metabolism, is generated by 25-hydroxyvitamin D 1α-hydroxylase in the kidney. Since 25-hydroxyvitamin D 1α-hydroxylase expression is enhanced by IRF-1, the present study explored the role of Chk2 for calcitriol formation and mineral metabolism. Chk2-deficient mice (chk2 (-/-)) were compared to wild-type mice (chk2 (+/+)). Transcript levels of renal 25-hydroxyvitamin D 1α-hydroxylase, Chk2, and IRF-1 were determined by RT-PCR; Klotho expression by Western blotting; bone density by µCT analysis; serum or plasma 1,25 (OH)2D3, PTH, and C-terminal FGF23 concentrations by immunoassays; and serum, fecal, and urinary calcium and phosphate concentrations by photometry. The renal expression of IRF-1 and 25-hydroxyvitamin D 1α-hydroxylase as well as serum 1,25 (OH)2D3 and FGF23 levels were significantly lower in chk2 (-/-) mice compared to chk2 (+/+) mice. Plasma PTH was not different between the genotypes. Renal calcium and phosphate excretion were significantly higher in chk2 (-/-) mice than in chk2 (+/+) mice despite hypophosphatemia and normocalcemia. Bone density was not different between the genotypes. We conclude that Chk2 regulates renal 25-hydroxyvitamin D 1α-hydroxylase expression thereby impacting on calcium and phosphate metabolism.

Regulation of mineral metabolism by lithium.

Lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), is widely used for the treatment of mood disorders. Side effects of lithium include nephrogenic diabetes insipidus, leading to renal water loss. Dehydration has in turn been shown to downregulate Klotho, which is required as co-receptor for the downregulation of 1,25(OH)2D3 formation by fibroblast growth factor 23 (FGF23). FGF23 decreases and 1,25(OH)2D3 stimulates renal tubular phosphate reabsorption. The present study explored whether lithium influences renal Klotho expression, FGF23 serum levels, 1,25(OH)2D3 formation, and renal phosphate excretion. To this end, mice were analyzed after a 14-day period of sham treatment or of treatment with lithium (200 mg/kg/day subcutaneously). Serum antidiuretic hormone (ADH), FGF23, and 1,25(OH)2D3 concentrations were determined by ELISA or EIA, renal Klotho protein abundance and GSK3 phosphorylation were analyzed by Western blotting, and serum phosphate and calcium concentration by photometry. Lithium treatment significantly increased renal GSK3 phosphorylation, enhanced serum ADH and FGF23 concentrations, downregulated renal Klotho expression, stimulated renal calcium and phosphate excretion, and decreased serum 1,25(OH)2D3 and phosphate concentrations. In conclusion, lithium treatment upregulates FGF23 formation, an effect paralleled by substantial decrease of serum 1,25(OH)2D3, and phosphate concentrations and thus possibly affecting tissue calcification.