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Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that plays a major role in the regulation of phosphate and vitamin D metabolism. FGF23 primarily acts on the kidneys to induce urinary phosphate excretion by downregulating the sodium-dependent phosphate cotransporters Napi2a and Napi2c and suppress 1,25-dihydroxyvitamin D [1,25(OH)2D] synthesis by altering the vitamin D-metabolizing enzymes CYP27b1 and CYP24a1 [1–3]. These functions of FGF23 are mediated by binding to FGF receptor 1 (FGFR1) and the transmembrane protein Klotho, which forms a specific receptor complex for FGF23 [4,5]. Importantly, recent observational studies have shown strong associations between elevated FGF23 and greater risk of cardiovascular events, particularly congestive heart failure, in patients with chronic kidney disease [6–8], hemodialysis patients [9,10], and individuals with apparently normal kidney function [11–15]. Furthermore, elevated FGF23 has been linked to volume overload, a common manifestation of heart failure [16–18]. As a potential mechanism of these observations, FGF23 is reported to induce cardiac hypertrophy via Klotho-independent signaling through the FGFR4 [19,20]. In addition, FGF23 increases renal sodium reabsorption by upregulating the Na+:Cl− cotransporter in the distal tubules via a Klotho-dependent pathway, leading to volume expansion, hypertension, and cardiac hypertrophy [21]. Interestingly, recent studies demonstrated an opposite directional relationship—injured cardiomyocyte could produce substantial amounts of FGF23 after myocardial infarction [22] or during cardiac hypertrophy [23]. However, it remains to be determined whether acute volume expansion, which induces hemodynamic congestion and cardiac overload, stimulates FGF23 production by cardiomyocytes. Here, we report our investigations into the impact of volume expansion by acute saline injection on cardiac and skeletal expressions of FGF23 as well as circulating FGF23 levels in rats.

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.

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.

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 SKF96365, 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.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.

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.

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.

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.

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 125(OH)2D3 formation by fibroblast growth factor 23 (FGF23). FGF23 decreases and 125(OH)2D3 stimulates renal tubular phosphate reabsorption. The present study explored whether lithium influences renal Klotho expression FGF23 serum levels 125(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 125(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 125(OH)2D3 and phosphate concentrations. In conclusion lithium treatment upregulates FGF23 formation an effect paralleled by substantial decrease of serum 125(OH)2D 3 and phosphate concentrations and thus possibly affecting tissue calcification. © 2013 Springer Verlag Berlin Heidelberg.

Besides their role in cardiac repolarization, human ether-a-go-go-related gene potassium (hERG) channels are expressed in several tumor cells including rhabdomyosarcoma cells. The channels foster cell proliferation. Ubiquitously expressed AMP-dependent protein kinase (AMPK) is a serine-/threonine kinase, stimulating energy-generating and inhibiting energy-consuming processes thereby helping cells survive periods of energy depletion. AMPK has previously been shown to regulate Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Ca²⁺ channels and K⁺ channels. The present study tested whether AMPK regulates hERG channel activity. Wild type AMPK (α1β1γ1), constitutively active (γR70Q)AMPK (α1β1γ1(R70Q)), or catalytically inactive (αK45R)AMPK (α1(K45R)β1γ1) were expressed in Xenopus oocytes with hERG. Tail currents were determined as a measure of hERG channel activity by two-electrode-voltage clamp. hERG membrane abundance was quantified by chemiluminescence and visualized by immunocytochemistry and confocal microscopy. Moreover, hERG currents were measured in RD rhabdomyosarcoma cells after pharmacological modification of AMPK activity using the patch clamp technique. Coexpression of wild-type AMPK and of constitutively active (γR70Q)AMPK significantly downregulated the tail currents in hERG-expressing Xenopus oocytes. Pharmacological activation of AMPK with AICAR or with phenformin inhibited hERG currents in Xenopus oocytes, an effect abrogated by AMPK inhibitor compound C. (γR70Q)AMPK enhanced the Nedd4-2-dependent downregulation of hERG currents. Coexpression of constitutively active (γR70Q)AMPK decreased membrane expression of hERG in Xenopus oocytes. Compound C significantly enhanced whereas AICAR tended to inhibit hERG currents in RD rhabdomyosarcoma cells. AMPK is a powerful regulator of hERG-mediated currents in both, Xenopus oocytes and RD rhabdomyosarcoma cells. AMPK-dependent regulation of hERG may be particularly relevant in cardiac hypertrophy and tumor growth.

Background/Aims: T-lymphocyte activation and function critically depends on Ca2+ signaling, which is regulated by store operated Ca2+ entry

// Shiwei Song 1 , Abul Fajol 2 , Xiankun Tu 1 , Baogang Ren 1 , Songsheng Shi 1 1 Department of Neurosurgery, Fujian Medical University Affiliated Union Hospital, Fujian Neurosurgical Institute, Fuzhou, 350001, China 2 Department of Clinical Sciences, CRC, Lund University, Malmo, 20502, Sweden Correspondence to: Shiwei Song, email: songshiweifmu@163.com Keywords: miR-204, GBM, suppressor Received: June 21, 2016 Accepted: August 16, 2016 Published: August 31, 2016 ABSTRACT 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

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.

To the Editor Depression is a multifaceted disorder with diverse causes and has been associated with the risk to develop severe medical disorders.1 Indeed, depression increases the risk of cardiovascular disease by 1.5-fold to 2-fold, of stroke by 1.8-fold, of Alzheimer disease by 2.1-fold, of diabetes by 60%, and of cancer by 1.3-fold to 1.8-fold.1 Fibroblast growth factors (FGFs) are best known for their regulatory roles in cell growth, differentiation, and morphogenesis in early stages of neural development and have been discussed as switch genes, biomarkers, and treatment targets for affective disorders recently.2,3 However, at least FGF-23 has also been proposed as a cardiovascular risk marker,4 a central player of disordered mineral metabolism,5 and acts to decrease phosphate, 1,25-dihydroxyvitamin D, and parathyroid hormone levels.5 A close, bidirectional relationship exists between depression and cardiovascular disease.1 Indeed, major depression is associated with an increased risk of coronary artery disease, myocardial infarction, congestive heart failure, and isolated systolic hypertension leading to increased mortality and morbidity in patients.1 Moreover, a strong relationship has been described between severe coronary and aortic calcifications, intima thickness, osteoporosis, and depressive disorders.1 Fibroblast growth factor 23 lowers serum levels of 1,25(OH)2D3, which in turn up-regulates renal and intestinal phosphate and calcium transport.6–9 In mice, it was shown recently that lithium treatment up-regulates FGF-23 formation, an effect paralleled by substantial decrease of serum 1,25(OH)2D3 and phosphate concentrations.10 The present study explores the effect of lithium treatment on serum FGF-23, 1,25(OH)2D3, calcium, and phosphate concentrations in depressed patients. A total of 95 acute depressive patients (age 48 ± 14 years) were recruited for this study. Inclusion criteria consist of unipolar depression, age older than 18 years, indication for antidepressant pharmacotherapy, insufficient response to an adequate antidepressant pretreatment and clinical indication for lithium augmentation, hamilton depression rating score greater than 12, and written informed consent. Diagnosis was confirmed on the basis of the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders. Fibroblast growth factor 23 serum concentrations were measured first in medicated patients before lithium augmentation and then after 4 weeks of medication with lithium. Detailed clinical data of the patients have already been published.11 All patients reached a lithium serum level of more than 0.4 mmol/L. Serum FGF-23 concentrations were measured by enzyme-linked immunosorbent assay (Immutopics International, California; AVP EIA kit, Phoenix Europe, Karlsruhe, Germany). enzyme-linked immunosorbent assay kits were employed to determine serum concentrations of 1,25(OH)2D3 (IDS, Boldon, United Kingdom). Data are provided as mean ± SEM; n represents the number of independent experiments. All data were tested for significance using unpaired Student t test. Only results with P < 0.05 were considered statistically significant. As illustrated in Figure ​Figure1,1, lithium treatment was followed by a marked increase of serum FGF-23 concentration. As shown in Figure ​Figure1,1, lithium treatment significantly decreased serum 1,25(OH)2D3 concentration. Lithium treatment significantly decreased serum phosphate concentrations (data not shown). FIGURE 1 Serum FGF-23 and 1,25(OH)2D3 levels/concentrations before and after lithium treatment. Arithmetic means ± SEM (n = 95) of serum FGF-23 (A) and 1,25(OH)2D3 levels (B) before (white bars) and after (black bars) lithium treatment. **P < 0.01 ...

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.

Glycogen synthase kinase (GSK)-3 is a ubiquitously expressed kinase inhibited by insulin-dependent Akt/PKB/SGK. Mice expressing Akt/PKB/SGK-resistant GSK3α/GSK3β (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 D(3) (1,25(OH)(2)D(3); 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)(2)D(3), 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 β-blocker propranolol. Urinary VMA excretion, serum FGF23, and renal phosphate and calcium excretion were significantly higher, and serum 1,25(OH)(2)D(3) 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)(2)D(3) formation, and thus mineral metabolism, by controlling the activity of the sympathetic nervous system.—Fajol, A., Chen, H., Umbach, A. T., Quarles, L. D., Lang, F., Föller, M. Enhanced FGF23 production in mice expressing PI3K-insensitive GSK3 is normalized by β-blocker treatment.

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.

Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) by renal 25-hydroxyvitamin D-3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)(2)D-3 regulates Ca2+ and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)(2)D-3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)(2)D-3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 mu M CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)(2)D-3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)(2)D-3 synthesis.

Contains fulltext : 108885.pdf (Publisher’s version ) (Open Access) Background: The oxidative stress-responsive kinase 1 (OSR1) participates in the WNK-(with no K) kinase dependent regulation of renal salt excretion and blood pressure. Little is known, however, about the role of OSR1 in the regulation of further renal transport systems. The present study analyzed the effect of OSR1 on NaPiIIa, the major renal tubular phosphate transporter. Methods: Immunohistochemistry and confocal microscopy were employed to determine renal localization of OSR1 and NaPiIIa. To elucidate the effect of OSR on NaPiIIa activity, cRNA encoding NaPiIIa was injected into Xenopus oocytes with or without additional injection of cRNA encoding OSR1, and phosphate transport was estimated from phosphateinduced currents determined with dual electrode voltage clamp. To elucidate the in vivo significance of OSR1 serum phosphate and hormone concentrations as well as urinary phosphate output of mice carrying one allele of WNK-resistant OSR1 (osr1tg/(+)) were compared to the respective values of wild type mice (osr1(+/+)). Results: NaPiIIa and OSR1 were both expressed in proximal renal tubule cells. Coexpression of OSR1 significantly up-regulated phosphate-induced currents in NaPiIIa-expressing Xenopus oocytes. Despite decreased serum phosphate concentration urinary phosphate excretion was significantly increased and NaPiIIa protein abundance in the brush border membrane significantly reduced in osr1tg/(+) mice as compared to osr1(+/+) mice. Serum PTH and calcitriol levels were similar in osr1tg/(+) mice and in osr1(+/+) mice, serum FGF23 concentration was, however, significantly higher in osr1tg/(+) mice than in osr1(+/+) mice. Conclusions: OSR1 is expressed in proximal renal tubules and participates in the regulation of FGF23 release and renal tubular phosphate transport.

The oxidative stress responsive kinase 1 (OSR1) contributes to WNK (with no K)-dependent regulation of renal tubular salt transport, renal salt excretion, and blood pressure. Little is known, however, about a role of OSR1 in the regulation of intestinal salt transport. The present study thus explored whether OSR1 is expressed in intestinal tissue and whether small intestinal Na+/H+ exchanger (NHE), small intestinal Na+-glucose cotransport (SGLT1), and/or colonic epithelium Na+ channel (ENaC) differ between knockin mice carrying one allele of WNK-resistant OSR1 ( osr1 +/KI) and wild-type mice ( osr1 +/+). OSR1 protein abundance was determined by Western blotting, cytosolic pH from BCECF fluorescence, NHE activity from Na+-dependent realkalinization following an ammonium pulse, SGLT1 activity from glucose-induced current, and colonic ENaC activity from amiloride-sensitive transepithelial current in Ussing chamber experiments. As a result, OSR1 protein was expressed in small intestine of both osr1 +/KI mice and osr1 +/+ mice. Daily fecal Na+, K+, and H2O excretion and jejunal SGLT1 activity were lower, whereas small intestinal NHE activity and colonic ENaC activity were higher in osr1 +/KI mice than in osr1 +/+ mice. NHE3 inhibitor S-3226 significantly reduced NHE activity in both genotypes but did not abrogate the difference between the genotypes. Plasma osmolarity, serum antidiuretic hormone, plasma aldosterone, and plasma corticosterone concentrations were similar in both genotypes. Small intestinal NHE3 and colonic α-ENaC protein abundance were not significantly different between genotypes, but colonic phospho-β-ENaC (ser633) was significantly higher in osr1 +/KI mice. In conclusion, OSR1 is expressed in intestine and partial WNK insensitivity of OSR1 increases intestinal NHE activity and colonic ENaC activity.

Apigenin, a flavone in fruits and vegetables, stimulates apoptosis and thus counteracts cancerogenesis. Erythrocytes may similarly undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)), ceramide formation and ATP depletion. The present study explored the effect of apigenin on eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, ceramide utilizing antibodies, and cytosolic ATP with luciferin-luciferase. A 48 h exposure to apigenin significantly increased [Ca(2+)](i) (≥ 1 μM), increased ceramide formation (15 μM), decreased ATP concentration (15 μM), decreased forward scatter (≥ 1 μM), and increased annexin V binding (≥ 5 μM) but did not significantly modify hemolysis. The effect of 15 μM apigenin on annexin V binding was blunted by Ca(2+) removal. The present observations reveal novel effects of apigenin, i.e. stimulation of Ca(2+) entry, ceramide formation and ATP depletion in erythrocytes with subsequent triggering of suicidal erythrocyte death, paralleled by cell shrinkage and phosphatidylserine exposure.

p38 protein kinase is activated by hyperosmotic shock, participates in the regulation of cell volume sensitive transport and metabolism and is involved in the regulation of various physiological functions including cell proliferation and apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include hyperosmotic shock, which increases cytosolic Ca(2+) activity and ceramide formation. The present study explored whether p38 kinase is expressed in human erythrocytes, is activated by hyperosmotic shock and participates in the regulation of eryptosis. Western blotting was utilized to determine phosphorylation of p38 kinase, forward scatter to estimate cell volume, annexin V binding to depict phosphatidylserine exposure and Fluo3 fluorescence to estimate cytosolic Ca(2+) activity. As a result, erythrocytes express p38 kinase, which is phosphorylated upon osmotic shock (+550 mM sucrose). Osmotic shock decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence, all effects significantly blunted by the p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM). In conclusion, p38 kinase is expressed in erythrocytes and participates in the machinery triggering eryptosis following hyperosmotic shock.

Klotho, a transmembrane protein, protease, and hormone mainly expressed in renal tissue counteracts aging. Overexpression of Klotho substantially prolongs the life span. Klotho deficiency leads to excessive formation of 1,25(OH)2D3, growth deficit, accelerated aging, and early death. Aging is frequently paralleled by dehydration, which is considered to accelerate the development of age-related disorders. The present study explored the possibility that dehydration influences Klotho expression. Klotho transcript levels were determined by RT-PCR, and Klotho protein abundance was detected by Western blotting in renal tissue from hydrated and 36-h-dehydrated mice as well as in human embryonic kidney (HEK293) cells. Dehydration was followed by a significant decline of renal Klotho transcript levels and protein abundance, accompanied by an increase in plasma osmolarity as well as plasma ADH, aldosterone, and 1,25(OH)2D3 levels. Antidiuretic hormone (ADH; 50 nM) and aldosterone (1 μM) significantly decreased Klotho transcription and protein expression in HEK293 cells. In conclusion, the present observations disclose a powerful effect of dehydration on Klotho expression, an effect at least partially mediated by enhanced release of ADH and aldosterone.

The present study was undertaken to evaluate the protective effect of turmeric powder on arsenic toxicity through mice model. Swiss albino male mice were divided into four groups. The first group was used as control, while groups 2, 3, and 4 were treated with turmeric powder (T, 50 mg/kg body weight/day), sodium arsenite (Sa, 10 mg/kg body weight/day) and turmeric plus Sa (T+Sa), respectively. Results showed that oral administration of Sa reduced the weight gain of the mice compared to the control group and food supplementation of turmeric prevented the reduction of weight gain. Turmeric abrogated the Sa-induced elevation of serum urea, glucose, triglyceride (TG) level and alanine aminotransferase (ALT) activity except the activity of alkaline phosphatase (ALP). Turmeric also prevented the Sa-induced perturbation of serum butyryl cholinesterase activity (BChE). Therefore, ameliorating effect of turmeric on Sa-treated mice suggested the future application of turmeric to reduce or to prevent arsenic toxicity in human.

Background Chronic arsenic exposure has been shown to cause liver damage. However, serum hepatic enzyme activity as recognized on liver function tests (LFTs) showing a dose-response relationship with arsenic exposure has not yet been clearly documented. The aim of our study was to investigate the dose-response relationship between arsenic exposure and major serum enzyme marker activity associated with LFTs in the population living in arsenic-endemic areas in Bangladesh. Methods A total of 200 residents living in arsenic-endemic areas in Bangladesh were selected as study subjects. Arsenic concentrations in the drinking water, hair and nails were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The study subjects were stratified into quartile groups as follows, based on concentrations of arsenic in the drinking water, as well as in subjects' hair and nails: lowest, low, medium and high. The serum hepatic enzyme activities of alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT) were then assayed. Results Arsenic concentrations in the subjects' hair and nails were positively correlated with arsenic levels in the drinking water. As regards the exposure-response relationship with arsenic in the drinking water, the respective activities of ALP, AST and ALT were found to be significantly increased in the high-exposure groups compared to the lowest-exposure groups before and after adjustments were made for different covariates. With internal exposure markers (arsenic in hair and nails), the ALP, AST and ALT activity profiles assumed a similar shape of dose-response relationship, with very few differences seen in the higher groups compared to the lowest group, most likely due to the temporalities of exposure metrics. Conclusions The present study demonstrated that arsenic concentrations in the drinking water were strongly correlated with arsenic concentrations in the subjects' hair and nails. Further, this study revealed a novel exposure- and dose- response relationship between arsenic exposure metrics and serum hepatic enzyme activity. Elevated serum hepatic enzyme activities in the higher exposure gradients provided new insights into arsenic-induced liver toxicity that might be helpful for the early prognosis of arsenic-induced liver diseases.

The present study was undertaken to evaluate the protective effect of turmeric powder on arsenic toxicity through mice model. Swiss albino male mice were divided into four groups. The first group was used as control, while groups 2, 3, and 4 were treated with turmeric powder (T, 50mg/kg body weight/day), sodium arsenite (Sa, 10mg/kg body weight/day) and turmeric plus Sa (T+Sa), respectively. Results showed that oral administration of Sa reduced the weight gain of the mice compared to the control group and food supplementation of turmeric prevented the reduction of weight gain. Turmeric abrogated the Sa-induced elevation of serum urea, glucose, triglyceride (TG) level and alanine aminotransferase (ALT) activity except the activity of alkaline phosphatase (ALP). Turmeric also prevented the Sa-induced perturbation of serum butyryl cholinesterase activity (BChE). Therefore, ameliorating effect of turmeric on Sa-treated mice suggested the future application of turmeric to reduce or to prevent arsenic toxicity in human. DOI: 10.3329/bmrcb.v36i3.7287Bangladesh Med Res Counc Bull 2010; 36: 82-88