Effects of dietary phosphorus concentration and phosphate salt form on renal tubule function in unilateral nephrectomized rats.

Background: Excessive consumption of phosphorus (P) impairs renal tubule function; however, the effects of different dietary phosphate salts on chronic kidney disease (CKD) are unclear. Aim: To examine the effects of potassium dihydrogen phosphate (KH2PO4) and potassium tripolyphosphate (K5P3O10) and P concentration on renal function in a rat model of early CKD. Methods: Male sham-operated Sprague-Dawley rats were fed a diet containing KH2PO4 with a normal P level. Kidney injury was induced by unilateral nephrectomy (UNx), and the rats were divided into four groups fed dietary KH2PO4 or K5P3O10 with a normal (UNx-NKH, UNx-NKP) or high (UNx-HKH, UNx-HKP) P concentration, respectively, for 21 days. Results: UNx-NKH rats showed significantly lower creatinine clearance (CCr) and higher albumin (ALB) compared with those of sham rats, confirming UNx-induced kidney injury. The urinary levels of liver-type fatty acid-binding protein (L-FABP) and ALB were significantly higher in UNx-HKP rats than in UNx-HKH rats. However, other markers of renal tubule function, such as CCr, serum creatinine (CRE), calcium (Ca), and hormones, only differed among groups according to the P concentration and not the dietary phosphate salt form. Histological examination showed higher incidence and severity of tubulointerstitial lesions, tubule regeneration, tubule dilation, and calcification in the high-phosphorus than in the normal-phosphorus UNx groups. These changes were more severe in the UNx-HKP group compared with the UNx-HKH group. Conclusion: This study highlights the importance of controlling dietary P intake in terms of both concentration and source to prevent the progression of CKD.

A short-term zinc-deficient diet decreases bone formation through down-regulated BMP2 in rat bone.

We investigated the effects of a short-term dietary zinc deficiency on bone metabolism. Zinc deficiency increased the mRNA expression of zinc uptake transporters such as Zip1, Zip13, and Zip14 in bone. However, zinc deficiency might not maintain zinc storage in bone, resulting in a decrease in bone formation through downregulation of the expression levels of osteoblastogenesis-related genes.

A combination of soy isoflavones and cello-oligosaccharides changes equol/O-desmethylangolensin production ratio and attenuates bone fragility in ovariectomized mice.

We examined the cooperative effects of isoflavones and cello-oligosaccharides on daidzein metabolism and bone fragility in ovariectomized mice. Cello-oligosaccharides increased urinary equol and decreased O-desmethylangolensin. A combination of isoflavones and cello-oligosaccharides attenuated decreases in bone breaking force and stiffness caused by ovariectomy. Combination treatment with isofalvones and cello-oligosaccharides increases urinary equol/O-desmethylangolensin production ratio and prevents ovariectomy-induced abnormalities in bone strength.

Dietary zinc deficiency induces oxidative stress and promotes tumor necrosis factor-α- and interleukin-1ß-induced RANKL expression in rat bone.

We investigated the effects of dietary zinc deficiency on oxidative stress and bone metabolism. Four-week-old male Wistar rats were randomly assigned to one of three groups for 4 weeks: a zinc-adequate group (30 ppm); a zinc-deficient group (1 ppm); and a pair-fed group (30 ppm) that was pair-fed to the zinc-deficient group. The iron content and the thiobarbituric acid reactive substance level in bone were higher in the zinc-deficient group than in the zinc-adequate and pair-fed groups. The mRNA expression level of osteoblastogenesis-related genes such as bone morphogenetic protein 2 and runt-related transcription factor 2 was lower in the zinc-deficient group than in the zinc-adequate and pair-fed groups. In contrast, the mRNA expression levels of tumor necrosis factor-α, interleukin-1ß and osteoclastogenesis-related genes such as receptor activator of nuclear factor-κB ligand and nuclear factor of activated T cells cytoplasmic 1 were higher in the zinc-deficient group than in the zinc-adequate and pair-fed groups. These findings suggested that dietary zinc deficiency reduced osteoblastogenesis via a decrease in the expression of bone morphogenetic protein 2 and increased osteoclastogenesis via enhancement of the expression of receptor for activator of nuclear factor-κB ligand induced by oxidative stress-stimulated tumor necrosis factor-α and interleukin-1ß.

Dietary zinc supplementation increased TNFα and IL1ß-induced RANKL expression, resulting in a decrease in bone mineral density in rats.

We investigated the effect of dietary zinc supplementation on bone metabolism in rats. Four-week-old male Wistar rats were fed a 30.0 mg zinc/kg diet (C), a 300.0 mg zinc/kg diet (HZ) or a 3,000.0 mg zinc/kg diet (EZ) for 4 weeks. The zinc content of the femur gradually increased in accordance with the gradual increase in the dietary zinc level. Although the mRNA expression of zinc transporters in bone did not differ between the groups, the mRNA expression of metallothioneins was increased in the HZ and EZ groups compared to the C group. Moreover, the bone mineral density was significantly decreased in the HZ and EZ groups compared to the C group. Furthermore, the mRNA expression of tumor necrosis factor α, Interleukin-1ß and osteoclastogenesis-related genes such as receptor for activator of nuclear factor-κB (NF-κB) ligand, tumor necrosis factor receptor-associated factor 6, and nuclear factor of activated T cells cytoplasmic 1 was significantly increased in the HZ and EZ groups compared to the C group. These findings suggested that dietary zinc supplementation reduced bone mineral density through the promotion of bone resorption via an increase in the expression of receptor for activator of NF-κB ligand induced by tumor necrosis factor α and Interleukin-1ß.

Activation of Nrf2/Keap1 signaling and autophagy induction against oxidative stress in heart in iron deficiency.

We investigated the effects of dietary iron deficiency on the redox system in the heart. Dietary iron deficiency increased heart weight and accumulation of carbonylated proteins. However, expression levels of heme oxygenase-1 and LC3-II, an antioxidant enzyme and an autophagic marker, respectively, in iron-deficient mice were upregulated compared to the control group, resulting in a surrogate phenomenon against oxidative stress.

Comparative activities of the S-enantiomer and racemic forms of equol on bone fragility in ovariectomized mice.

We compared the effects of the S-enantiomer and racemic forms of equol on bone using ovariectomized (OVX) mice. Femoral bone mineral density and bone strength decreased in the OVX mice, but not in OVX mice administered 0.5 mg/d S-equol. This, however, did not hold for racemic equol. Serum and urine S-equol concentrations were higher in the mice administered S-equol than in those administered racemic equol. These results suggest that the inhibitory effects of S-equol on bone fragility in OVX mice are greater than those of racemic equol.

Iron deficiency negatively regulates protein methylation via the downregulation of protein arginine methyltransferase.

Iron is an essential trace metal for all biological processes and plays a role in almost every aspect of body growth. Previously, we found that iron-depletion downregulated the expression of proteins, arginine methyltransferase-1 and 3 (PRMT1 and PRMT3), by an iron-specific chelator, deferoxamine (DFO), in rat liver FAO cell line using DNA microarray analysis (unpublished data). However, regulatory mechanisms underlying the association between iron deficiency and PRMT expression are unclear in vitro and in vivo. In the present study, we revealed that the treatment of cells with two iron-specific chelators, DFO and deferasirox (DFX), downregulated the gene and protein expression of PRMT1 and 3 as compared with the untreated cells. Subsequently, DFO and DFX treatments decreased protein methylation. Importantly, these effects were attenuated by a holo-transferrin treatment. Furthermore, weanling Wistar-strain rats were fed a control diet or an iron-deficient diet for 4 weeks. Dietary iron deficiency was found to decrease the concentration of hemoglobin and liver iron while increasing the heart weight. PRMT and protein methylation levels were also significantly reduced in the iron-deficient group as compared to the control group. To our knowledge, this is the first study to demonstrate that PRMT levels and protein methylation are reduced in iron-deficient models, in vitro and in vivo.

Severe iron deficiency decreases both bone formation and bone resorption in rats.

The purpose of this study was to clarify the manner in which dietary iron deficiency decreased bone mineral density (BMD) in rats. Eighteen 3-wk-old male Wistar rats were divided into 3 groups of 6 rats each. The rats in 2 of the 3 groups had free access to a control diet (C group) or an iron-deficient diet (ID group) for 4 wk. The rats in the third group (PF group) were pair-fed the control diet to the mean intake of the ID group. Compared with the C and PF groups, hematocrit and hemoglobin concentrations were significantly reduced and bone mineral content and BMD of the femur were significantly lower in the ID group. Bone histomorphometric parameters showed that the bone formation rate and osteoclast surface in the lumbar vertebra were significantly reduced in the ID group compared with the C and PF groups. Furthermore, dietary iron deficiency decreased serum 1,25-dihydroxycholecalciferol, insulin-like growth factor-I, and osteocalcin concentrations and urinary excretion of deoxypyridinoline. These results suggest that severe iron deficiency decreases not only bone formation but also bone resorption.

Hypoglycemic and hypolipidemic effects of hesperidin and cyclodextrin-clathrated hesperetin in Goto-Kakizaki rats with type 2 diabetes.

We investigated the hypoglycemic and hypolipidemic effects of two hesperertin glycosides, namely, hesperidin and cyclodextrin (CD)-clathrated hesperetin, in Goto-Kakizaki (GK) weanling rats with type 2 diabetes. We demonstrated that hesperidin and CD-hesperetin normalized glucose metabolism by altering the activities of glucose-regulating enzymes and reducing the levels of lipids in the serum and liver of the GK rats. These effects of hesperidin glycosides were partly produced by altering the expression of genes encoding the peroxisome proliferator-activated receptors, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, and the low-density lipoprotein receptor.

Distribution and major sources of flavonoid intakes in the middle-aged Japanese women.

We estimated the intake of individual flavonoids in a cross sectional study and clarified the major sources contributing to the flavonoid levels in the middle-aged Japanese women by a 24-h weighed dietary record study. The subjects included in the study were 516 free-living women. Each subject completed a 24-h weighed dietary record and received a health check-up. We used the Functional Food Factor database for estimating the intake of 5 major flavonoid intakes, i.e. flavan-3-ols, isoflavones, flavonols, flavanones and flavones. The mean intake of flavan-3-ols, isoflavones, flavonols, flavanones and flavones was 1277, 216, 58, 31 and 15 micromol/d, respectively. The richest source of flavan-3-ols was green tea. The 3 major food sources of isoflavone were the processed soy foods and those of flavonol were the onion, moroheiya (nalta jute) and Japanese radish leaves. Grapefruit and citrus fruit juices were the major sources of flavanones, and tsurumurasaki (malabar spinach), green pepper and grapefruit were the main sources of flavone. Furthermore, analysis of sub-samples from middle-aged Japanese women indicated that there may be an association between flavonoid intake and the levels of oxidized LDL, which might be related to the incidence of cardiovascular diseases.

Comparative activities of daidzein metabolites, equol and O-desmethylangolensin, on bone mineral density and lipid metabolism in ovariectomized mice and in osteoclast cell cultures.

Daidzein, a major isoflavone predominantly found in soybean, is mainly metabolized to equol and O-desmethylangolensin (O-DMA) by the human gut microflora. Equol exhibits a stronger estrogenic activity than daidzein, however, only approximately 30% of the population has been identified as equol-producers and there are too few direct evidences of the effects of the other major metabolite, O-DMA on estrogen-deficient status. The purpose of this study is therefore, to compare the effect of both O-DMA and equol on bone and lipid metabolism in vivo and in vitro. For the in vivo study, 8-week-old female mice were assigned to five groups as follows: sham-operated (sham), ovariectomized (OVX), OVX + 0.5 mg/day O-DMA (OVX + O-DMA), OVX + 0.5 mg/day equol (OVX + Eq), and OVX + 0.03 microg/day 17beta-estradiol (OVX + E2) administration. Three weeks after the intervention, O-DMA and equol did not affect uterine atrophy in OVX mice. The bone mineral density (BMD) of the femur was lower in the OVX group than in the sham group. The administration of equol but not O-DMA, maintained BMD through the intervention. Values of whole body fat mass and plasma lipids were lower in the equol and O-DMA treated OVX mice than those in OVX mice. In the in vitro study, equol significantly inhibited the osteoclast formation induced by 1alpha,25(OH)(2)D(3) in a dose-dependent manner in a co-culture system of mouse bone-marrow cells with primary osteoblastic cells. However, O-DMA slightly inhibited osteoclast formation, and the effect was not dose dependent. These results suggest that the effects of O-DMA on bone and lipid metabolism in OVX mice and osteoclast cell cultures are weaker than those of equol.

Effects of dietary ascorbic acid supplementation on lipid peroxidation and the lipid content in the liver and serum of magnesium-deficient rats.

We investigated the effects of ascorbic acid (AsA) supplementation on lipid peroxidation and the lipid content in the liver and serum of magnesium (Mg)-deficient rats. Eighteen 3-week-old male Sprague-Dawley strain rats were divided into 3 groups and maintained on a control diet (C group), a low-Mg diet (D group), or a low-Mg diet supplemented with AsA (DA group) for 42 d. At the end of this period, the final body weight, weight gain, and serum Mg concentrations were significantly decreased in the Mg-deficient rats. Further, dietary AsA supplementation had no effect on the growth, serum Mg concentration, Mg absorption, and Mg retention. The serum concentration of AsA was significantly lower in the D group than in the C group but was unaltered in the DA group. The levels of phosphatidylcholine hydroperoxide (PCOOH) in the serum and of triglycerides (TGs) and total cholesterol (TC) in the serum and liver were significantly higher in the D group than in the C group. The serum PCOOH, liver TG, and liver TC levels were decreased in the DA group. These results indicate that Mg deficiency increases the AsA requirement of the body and that AsA supplementation normalizes the serum levels of PCOOH and the liver lipid content in Mg-deficient rats, without altering the Mg status.

Citrus nobiletin suppresses bone loss in ovariectomized ddY mice and collagen-induced arthritis in DBA/1J mice: possible involvement of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis regulation.

Bone resorption is known to accelerate during the onset of several disorders, including osteoporosis (OP) and rheumatoid arthritis (RA). Some epidemiological surveys have suggested that a high intake of vegetables and fruits has an inverse relation to such disease incidence, though the number of active constituents elucidated thus far is limited. In the present study, we examined the efficacy of various food phytochemicals using two animal models. First, female ddY mice were ovariectomized (OVX) or sham-operated (sham), after which five different compounds (phenethyl isothiocyanate, zerumbone, auraptene, 1'-acetoxychavicol acetate, and nobiletin) were administered separately to OVX mice with a mini-osmotic pump at doses of 0.25 or 0.5 mg/day for 4 weeks, with 17beta-estradiol (E_{2}, 0.03 microg/day) used as a positive control. Nobiletin, in contrast to the other tested phytochemicals, significantly (P<0.05) suppressed the reduction of whole bone mineral density by 61%, which was comparable to or higher than the efficacy of E_{2}. Next, nobiletin given as an i.p. administration at 20 mg/kg of body weight, but not 2 mg/kg, to male DBA/1J mice every 2 days for 12 days led to a marked decrease in type II collagen-induced arthritis by 45% (P < 0.05). Furthermore, the flavonoid (4-50 microM) attenuated receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis of RAW264.7 cells, as detected by tartarate-resistant acid phosphatase activity and microscopic observations. Of note, nobiletin also suppressed RANKL-activated extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2, and p38 mitogen-activated protein kinase activities, and thereby regulated the promoter activation of nuclear factor kappaB (NFkappaB) and activator protein-1, key transcription factors for differentiation. Together, our results suggest that nobiletin is a promising phytochemical for the prevention or treatment of osteoclastogenesis-related disorders, including OP and RA, with reasonable action mechanisms.

Effects of Oral Administration of Moringa oleifera Lam on Glucose Tolerance in Goto-Kakizaki and Wistar Rats.

Medicinal plants constitute an important source of potential therapeutic agents for diabetes. In the present study, we investigated the effects of Moringa oleifera (MO) Lam, Moringacea, on glucose tolerance in Wistar rats and Goto-Kakizaki (GK) rats, modeled type 2 diabetes. Major polyphenols in MO powder were quercetin glucosides, rutin, kaempferol glycosides and chlorogenic acids by HPLC analysis. As the results of glucose tolerance test, MO significantly decreased the blood glucose at 20, 30, 45and 60 min for GK rats and at 10, 30 and 45 min for Wistar rats (p<0.05) compared to the both controls after glucose administration. The area under the curve of changes in the blood glucose was significantly higher in the GK control group than in the GK plus MO group (p<0.05) in the periods 30-60 min and 60-120 min. Furthermore, MO significantly decreased stomach emptying in GK rats (p<0.05). The results indicated that MO has an ameliorating effect for glucose intolerance, and the effect might be mediated by quercetin-3-glucoside and fiber contents in MO leaf powder. The action of MO was greater in GK rats than in Wistar rats.

High-phosphorus diet induces osteopontin expression of renal tubules in rats.

High-phosphorus (P) diet induces nephrocalcinosis in rats; however, the mechanism for onset of this disorder is unclear. The calcium (Ca) deposits in kidney are a form of hydroxyapatite, while osteopontin is combined with hydroxyapatite. Based on these observations, we speculated that the osteopontin play an important role in the formation of the Ca deposits induced by high-P diet. This study was investigated the effect of high-P diet on osteopontin expression in kidney. Female Wistar rats were fed diets containing P concentrations of either 0.3% (control diet) or 1.5% (high-P diet) for 14 days. On von Kossa staining, Ca deposits were seen in the tubules of the cortex, outer medulla and inner medulla in rats fed on the high-P diet. Expression of osteopontin was confirmed in rats fed on the high-P diet by immunohistochemical staining, and the localization of this protein was in the same region as the Ca deposits. On the other hand, no evidence of Ca deposits and osteopontin expression was observed in the tubules of the cortex, outer medulla or inner medulla of rats fed on the control diet. These results suggest that high-P diet induces osteopontin expression in the renal tubules. Moreover, our results suggest that increase in osteopontin expression in the renal tubules is presumably involved in the formation of Ca deposits induced by high-P diet.

Iron deficiency induces autophagy and activates Nrf2 signal through modulating p62/SQSTM.

Iron is an essential trace metal in almost all organisms and plays an important role in the redox system. We previously reported that iron deficiency activated autophagy and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling for oxidative stress. However, regulatory mechanisms underlying the association between autophagy and Nrf2 signaling are unclear. In this study, we found that treatment of cells with an iron-specific chelator deferoxamine (DFO) increased reactive oxidative species (ROS) production by elevating the expression of p47phox and p67phox compared with that in untreated cells. The DFO treatment also induced protein aggregation and formed aggresome, which is a cellular response to misfolded protein. In addition, DFO treatment upregulated the expression of the autophagic gene p62/SQSTM1, which in turn activated intracellular proteolysis during autophagy. DFO treatment phosphorylated p62/SQSTM1 (Thr351) to activate Nrf2. However, silencing of p62/SQSTM1 followed by DFO treatment attenuated Nrf2 activation and resulted in the accumulation of carboxyl proteins compared with DFO treatment alone. These results indicated that iron deficiency activates Nrf2 signaling by modulating p62/SQSTM1 during autophagy.

A case of empyema and septic arthritis due to Nocardia farcinica.

Septic arthritis due to Nocardia sp. should be suspected when a patient with risk factors such as pneumoconiosis or diabetes mellitus develops joint symptoms, especially if the patient has had nocardiosis in other sites.

Dietary fibers from mushroom sclerotia. 4. In vivo mineral absorption using ovariectomized rat model.

The effect of three novel dietary fibers (DFs) prepared from mushroom sclerotia, namely, Pleurotus tuber-regium, Polyporus rhinocerus, and Wolfiporia cocos, on calcium and magnesium absorption was evaluated in ovariectomized (OVX) rats fed with sclerotial DF based and low Ca (0.3%) diets for 14 days. The animals in the W. cocos DF diet group possessed significantly (p < 0.05) higher levels of cecal total short-chain fatty acids (204 mumol/g of cecal content) and had an acidic pH (5.88) in their cecum when compared with those of the cellulose control group. Such an acidic environment was found to promote the ionization of the unabsorbed Ca and Mg in their cecum, which in turn significantly (p < 0.05) increased the concentrations of cecal soluble Ca (2.56-fold) and Mg (1.22-fold). Besides, the apparent Ca and Mg absorptions of the W. cocos DF group were also significantly (p < 0.05) enhanced (Ca, 16.5%; Mg, 15.3%) together with significantly (p < 0.05) higher serum Ca (3.61 mmol/L) and Mg (1.07 mmol/L) levels when compared with those of the cellulose control group. These data suggest that ingestion of W. cocos DF could improve the overall Ca and Mg absorptions of the OVX rats fed a low Ca diet. The potential use of sclerotial DFs as a functional food ingredient for enhancing mineral absorption is also discussed.

Kanamycin inhibits daidzein metabolism and abilities of the metabolites to prevent bone loss in ovariectomized mice.

BACKGROUND: Daidzein is an isoflavone derived from soybeans that exerts preventive effects on bone loss in ovariectomized (OVX) animals. These effects have been correlated with increasing serum equol levels. In the present study, we investigated the effects of antibiotic intake on equol metabolism from daidzein, and the corresponding levels of bone loss in OVX mice. METHODS: Eight-week-old female ddY mice (n = 42) were either ovariectomized (OVX) or subjected to a sham operation (sham). OVX mice were then divided into six dietary subgroups: control diet (control), 0.3 % kanamycin diet (KN), 0.1 % daidzein diet (Dz), 0.1 % daidzein and 0.0375 % kanamycin diet (Dz+KN3.75), 0.1 % daidzein and 0.075 % kanamycin diet (Dz+KN7.5), and 0.1 % daidzein and 0.3 % kanamycin diet (Dz+KN30). The mice were fed their respective diets for 4 weeks. RESULTS: Uterine weight and femoral bone mineral density (BMD) were significantly lower in the OVX mice compared in the sham mice. No significant differences in uterine weight were observed among all OVX dietary subgroups. The Dz subgroup was found to exhibit higher plasma equol and O-desmethylangolensin (O-DMA) concentrations, as well as greater femoral BMD, compared to all other OVX subgroups. Furthermore, when compared to the Dz group, kanamycin intake decreased plasma equol and O-DMA concentrations, as well as femoral BMD in the OVX mice. CONCLUSIONS: These results suggest that kanamycin intake inhibited the conversion of daidzein to equol and O-DMA, blocking the preventive effects of daidzein on bone loss in OVX mice. Therefore, the bone-protective effects of daidzein intake may be predominantly associated with increased plasma concentrations of either equol or O-DMA.