Functional kupffer cells migrate to the liver from the intraperitoneal cavity.

We established a method of KC transplantation by intraperitoneal (i.p.) injection using EGFP-expressing cells (EGFP-KCs) and normal KCs. The novel method is easier and less invasive than conventional methods so that it is not only technically advantageous but also ethically preferable for experiments using animals. We demonstrated that KCs migrated to the liver following i.p. Injection. Engraftment in the liver was not observed for peritoneal macrophages (pMPs). This suggests that KCs migrate to the liver via a sorting mechanism. KC injection decreased the KC number at 24 h and then recovered the KCs at 10 days to a normal level. Additionally, recovery to the normal level by KC injection was observed in mice with KC depletion induced by GdCl3. These results suggest that a regulatory mechanism exists for controlling the number of KCs.

All-trans retinoic acid reduces the transcriptional regulation of intestinal sodium-dependent phosphate co-transporter gene (Npt2b).

Inorganic phosphate (Pi) homeostasis is regulated by intestinal absorption via type II sodium-dependent co-transporter (Npt2b) and by renal reabsorption via Npt2a and Npt2c. Although we previously reported that vitamin A-deficient (VAD) rats had increased urine Pi excretion through the decreased renal expression of Npt2a and Npt2c, the effect of vitamin A on the intestinal Npt2b expression remains unclear. In this study, we investigated the effects of treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, on the Pi absorption and the Npt2b expression in the intestine of VAD rats, as well as and the underlying molecular mechanisms. In VAD rats, the intestinal Pi uptake activity and the expression of Npt2b were increased, but were reduced by the administration of ATRA. The transcriptional activity of reporter plasmid containing the promoter region of the rat Npt2b gene was reduced by ATRA in NIH3T3 cells overexpressing retinoic acid receptor (RAR) and retinoid X receptor (RXR). On the other hand, CCAAT/enhancer-binding proteins (C/EBP) induced transcriptional activity of the Npt2b gene. Knockdown of the C/EBP gene and a mutation analysis of the C/EBP responsible element in the Npt2b gene promoter indicated that C/EBP plays a pivotal role in the regulation of Npt2b gene transcriptional activity by ATRA. EMSA revealed that the RAR/RXR complex inhibits binding of C/EBP to Npt2b gene promoter. Together, these results suggest that ATRA may reduce the intestinal Pi uptake by preventing C/EBP activation of the intestinal Npt2b gene.

Association of increased renal Cyp24a1 gene expression with low plasma 1,25-dihydroxyvitamin D levels in rats with streptozotocin-induced diabetes.

Decreases in plasma vitamin D concentrations have been reported in diabetes, although the mechanism involved in this decrease is unclear. Here, we investigated the association between Cyp24a1, a vitamin D catabolic enzyme, and abnormalities in vitamin D metabolism in streptozotocin-induced diabetes rats, an animal model of type 1 diabetes. Plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] levels were significantly lower in streptozotocin-induced diabetes rats and renal Cyp24a1 mRNA expression levels were increased. Western blotting analysis of streptozotocin-induced diabetes rats kidney tissues with anti-CYP24A1 antibody showed a strong signal around 40 kDa, which differs from the predicted 50-55 kDa molecular weight for full-length Cyp24a1 and could represent the Cyp24a1-splicing variant that lacks exons 1 and 2. We observed high levels of renal Cyp24a1-splicing variant mRNA expression in streptozotocin-induced diabetes rats. We also confirmed transcriptional up-regulation of endogenous Cyp24a1 mRNA expression through glucocorticoid receptors by glucocorticoid in opossum kidney proximal cells. Taken together, our results indicated that high Cyp24a1 expression levels may play a role in the decrease of plasma 1,25(OH)2D levels in streptozotocin-induced diabetes rats. High plasma corticosterone levels in diabetes may affect transcriptional regulation to promote increases in Cyp24a1 expression.

Regulation of α-Klotho Expression by Dietary Phosphate During Growth Periods.

Inorganic phosphate (Pi) is an essential nutrient for maintaining various biological functions, particularly during growth periods. Excess intake of dietary Pi increases the secretion of fibroblast growth factor 23 (FGF23) and parathyroid hormone to maintain plasma Pi levels. FGF23 is a potent phosphaturic factor that binds to the α-klotho/FGFR complex in the kidney to promote excretion of Pi into the urine. In addition, excess intake of dietary Pi decreases renal α-klotho expression. Down-regulation or lack of α-klotho induces a premature aging-like phenotype, resulting from hyperphosphatemia, and leading to conditions such as ectopic calcification and osteoporosis. However, it remains unclear what effects dietary Pi has on α-klotho expression at different life stages, especially during growth periods. To investigate this, we used C57BL/6J mice in two life stages during growing period. Weaned (3 weeks old) and periadolescent (7 weeks old) were randomly divided into seven experimental groups and fed with 0.02, 0.3, 0.6, 0.9, 1.2, 1.5, or 1.8% Pi diets for 7 days. As a result, elevated plasma Pi and FGF23 levels and decreased renal α-klotho expression were observed in weaned mice fed with a high Pi diet. In addition, a high Pi diet clearly induced renal calcification in the weaned mice. However, in the periadolescent group, renal calcification was not observed, even in the 1.8% Pi diet group. The present study indicates that a high Pi diet in weaned mice has much greater adverse effects on renal α-klotho expression and pathogenesis of renal calcification compared with periadolescent mice.

Sterol regulatory element binding protein 1 trans-activates 25-hydroxy vitamin D3 24-hydroxylase gene expression in renal proximal tubular cells.

The physiological activity of the steroid derived hormone vitamin D is regulated by several enzymatic steps. Both 25-hydroxy vitamin D3 1α-hydroxylase (CYP27B1) and 25-hydroxyvitamin D3 24-hydroxylase (CYP24A1) modulate blood levels of 1,25-dihydroxyvitamin D3, an activated form of vitamin D. We previously demonstrated that CYP27B1 expression was trans-activated by sterol regulatory element binding protein 1 (SREBP1), although whether SREBP1 also regulates CYP24A1 transcription was unclear. Here we investigated the ability of SREBP1 to affect CYP24A1 transcription. In a luciferase reporter assay, mouse and human CYP24A1 promoter activity was strongly activated by SREBP1 in opossum kidney proximal tubular cells (OK-P). Three putative SREs (pSREs) were found in the mouse Cyp24a1 gene promoter and the SREBP1 protein showed specific binding to the pSRE1 element in EMSAs. Site-directed mutagenesis of the pSRE1 element strongly decreased SREBP1-mediated Cyp24a1 gene transcription. Moreover, siRNA-mediated SREBP1 knock-down repressed CYP24A1 expression in human renal proximal tubular epithelial cells (HKC-8). In animal studies, mice given various doses of thyroid hormone (T3) showed dose-dependent reductions in renal Srebp1c and Cyp24a1 mRNA levels. Taken together, our results suggest that SREBP1 trans-activates CYP24A1 expression through SREBP binding elements present in the promoter.

The age-related changes of dietary phosphate responsiveness in plasma 1,25-dihydroxyvitamin D levels and renal Cyp27b1 and Cyp24a1 gene expression is associated with renal α-Klotho gene expression in mice.

In this study, we investigated the relationship between age-related changes in renal α-Klotho gene expression, vitamin D metabolism and the responsiveness of dietary phosphate in 1, 2 and 13 month-old mice fed a high phosphate (phosphate 1.2%) diet or low phosphate (phosphate 0.02%) diet for 5 days. We found that 1,25-dihydroxyvitamin D levels in plasma were significantly lower in the high phosphate group than the low phosphate group for 1 and 2 month-old mice, but not 13 month-old mice. In addition, in the high phosphate group plasma 1,25-dihydroxyvitamin D levels were decreased in 2 month-old mice relative to 1 month-old mice, but 13 month-old mice had higher levels than 2 month-old mice. In fact, plasma 1,25-dihydroxyvitamin D levels showed a significant correlation with vitamin D metabolism gene Cyp27b1 and Cyp24a1 mRNA expression in the high phosphate group. Interestingly, renal α-Klotho mRNA and protein levels were significant change with age. Furthermore, α-Klotho mRNA expression showed a significant negative correlation with plasma 1,25-dihydroxyvitamin D levels in the high phosphate group. Our results suggest that age-related alterations in renal α-Klotho expression could affect the responsiveness of dietary phosphate to vitamin D metabolism.

Dietary phosphate supplementation delays the onset of iron deficiency anemia and affects iron status in rats.

Inorganic phosphate (Pi) plays critical roles in bone metabolism and is an essential component of 2,3-diphosphoglycerate (2,3-DPG). It has been reported that animals fed a low-iron diet modulate Pi metabolism, whereas the effect of dietary Pi on iron metabolism, particularly in iron deficiency anemia (IDA), is not fully understood. In this study, we hypothesized the presence of a link between Pi and iron metabolism and tested the hypothesis by investigating the effects of dietary Pi on iron status and IDA. Wistar rats aged 4 weeks were randomly assigned to 1 of 4 experimental dietary groups: normal iron content (Con Fe)+0.5% Pi, low-iron (Low Fe)+0.5% Pi, Con Fe+1.5% Pi, and Low Fe+1.5% Pi. Rats fed the 1.5% Pi diet for 14 days, but not for 28 days, maintained their anemia state and plasma erythropoietin concentrations within the reference range, even under conditions of low iron. In addition, plasma concentrations of 2,3-DPG were significantly increased by the 1.5% Pi diets and were positively correlated with plasma Pi concentration (r=0.779; P<.001). Dietary Pi regulated the messenger RNA expression of iron-regulated genes, including divalent metal transporter 1, duodenal cytochrome B, and hepcidin. Furthermore, iron concentration in liver tissues was increased by the 1.5% Pi in Con Fe diet. These results suggest that dietary Pi supplementation delays the onset of IDA and increases plasma 2,3-DPG concentration, followed by modulation of the expression of iron-regulated genes.

Short-term dietary phosphate restriction up-regulates ileal fibroblast growth factor 15 gene expression in mice.

Members of the fibroblast growth factor (FGF) 19 subfamily, including FGF23, FGF15/19, and FGF21, have a role as endocrine factors which influence the metabolism of inorganic phosphate (Pi) and vitamin D, bile acid, and energy. It has been reported that dietary Pi regulates circulating FGF23. In this study, the short-term effects of dietary Pi restriction on the expression of FGF19 subfamily members in mice were analyzed. An initial analysis confirmed plasma FGF23 levels positively correlated with the amount of dietary Pi. On the other hand, ileal Fgf15 gene expression, but not hepatic Fgf21 gene expression, was up-regulated by dietary Pi restriction. In addition, we observed the increase of plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] levels by dietary Pi restriction, and the up-regulation of ileal Fgf15 mRNA expression by 1,25(OH)2D3 and vitamin D receptor (VDR). Importantly, dietary Pi restriction-induced Fgf15 gene expression was prevented in VDR-knockout mice. Furthermore, diurnal variations of plasma triglyceride concentrations and hepatic mRNA expression of the bile acid synthesis enzyme Cyp7a1 as one of Fgf15 negative target genes was influenced by dietary Pi restriction. These results suggest that dietary Pi restriction up-regulates ileal Fgf15 gene expression through 1,25(OH)2D3 and VDR, and may affect hepatic bile acid homeostasis.

Downregulation of renal type IIa sodium-dependent phosphate cotransporter during lipopolysaccharide-induced acute inflammation.

The type IIa sodium-dependent phosphate cotransporter (Npt2a) plays a critical role in reabsorption of inorganic phosphate (Pi) by renal proximal tubular cells. Pi abnormalities during early stages of sepsis have been reported, but the mechanisms regulating Pi homeostasis during acute inflammation are poorly understood. We examined the regulation of Pi metabolism and renal Npt2a expression during lipopolysaccharide (LPS)-induced inflammation in mice. Dose-response and time-course studies with LPS showed significant increases of plasma Pi and intact parathyroid hormone (iPTH) levels and renal Pi excretion, while renal calcium excretion was significantly decreased. There was no difference in plasma 1,25-dihydroxyvitamin D levels, but the induction of plasma intact fibroblast growth factor 23 levels peaked 3 h after LPS treatment. Western blotting, immunostaining, and quantitative real-time PCR showed that LPS administration significantly decreased Npt2a protein expression in the brush border membrane (BBM) 3 h after injection, but there was no change in renal Npt2a mRNA levels. Moreover, tumor necrosis factor-α injection also increased plasma iPTH and decreased renal BBM Npt2a expression. Importantly, we revealed that parathyroidectomized rats had impaired renal Pi excretion and BBM Npt2a expression in response to LPS. These results suggest that the downregulation of Npt2a expression in renal BBM through induction of plasma iPTH levels alter Pi homeostasis during LPS-induced acute inflammation.

Hypercholesterolemia and effects of high cholesterol diet in type IIa sodium-dependent phosphate co-transporter (Npt2a) deficient mice.

The type IIa sodium-dependent phosphate co-transporter (Npt2a) is important to maintain renal inorganic phosphate (Pi) homeostasis and the plasma Pi levels. It has reported that disorder of Pi metabolism in kidney can be risk factors for cardiovascular disease as well as hypercholesterolemia. However, the relationship between Pi and cholesterol metabolism has not been clarified. The current study investigated the effects of Npt2a gene ablation that is known as hypophosphatemia model on cholesterol metabolism in mice. Npt2a deficient (Npt2a(-/-)) mice and wild type mice were fed diets with or without 2% cholesterol for 12 days. Plasma lipid and lipoprotein profile analysis revealed that plasma lipid levels (total, LDL and HDL cholesterol) were significantly higher in Npt2a(-/-) mice than wild type (WT) mice. Interestingly, high cholesterol diet markedly increased plasma levels of total, LDL and HDL cholesterol in WT mice, but not Npt2a(-/-) mice. On the other hand, there were no differences in body and liver weight, intake and hepatic lipid accumulation between WT and Npt2a(-/-) mice. These results suggest that ablation of Npt2a gene induces hypercholesterolemia and affects the ability to respond normally to dietary cholesterol.

Dietary phosphate restriction induces hepatic lipid accumulation through dysregulation of cholesterol metabolism in mice.

Excessive inorganic phosphate (Pi) intake and hyperphosphatemia have both been speculated to be risk factors for cardiovascular disease and hypercholesterolemia, and dysregulation of cholesterol metabolism can lead to atherosclerosis. However, the relationship between Pi and cholesterol metabolism has not been investigated in detail. Our recent study showed that triiodothyronine can induce both hyperphosphatemia and hypocholesterolemia in mice. We therefore hypothesized a possible linkage between Pi and cholesterol metabolism. In this study, we investigated the effects of dietary Pi intake on cholesterol metabolism in mice. Mice were divided into 4 groups, which were fed diets containing 1.2% or 0.1% Pi and with or without 2% cholesterol (Pi-sufficient, Pi-restricted, Pi-sufficient + Chol, and Pi-restricted + Chol), for 12 days. Inorganic phosphate-restricted mice exhibited significantly higher liver weights than did Pi-sufficient mice. Interestingly, dietary Pi restriction significantly increased high-cholesterol diet-induced hepatic lipid accumulation. Real-time polymerase chain reaction analysis revealed that dietary Pi restriction decreased expression of hepatic genes involved in cholesterol metabolism and fatty acid biosynthesis. In addition, hepatic messenger RNA levels of several transcription factors including peroxisome proliferator-activated receptors and liver X receptor were markedly decreased by Pi restriction. Furthermore, plasma lipid and lipoprotein profile analysis showed that dietary Pi restriction reduced susceptibility to high-cholesterol diet-induced hyperlipidemia. Importantly, we found that there was a significant negative correlation between plasma levels of Pi and total cholesterol. These results suggest that dietary Pi plays an important role in the development of fatty liver disease and hyperlipidemia induced by a high-cholesterol diet through regulation of lipid metabolism-related gene expression in the liver.

Thyroid hormones decrease plasma 1α,25-dihydroxyvitamin D levels through transcriptional repression of the renal 25-hydroxyvitamin D3 1α-hydroxylase gene (CYP27B1).

The primary determinant of circulating 1α,25-dihydroxyvitamin D (1,25[OH](2)D) levels is the activity of 25-hydroxyvitamin D-1α-hydroxylase (cytochrome P450 27B1 [CYP27B1]) in the kidney. Hyperthyroid patients have been reported to have low levels of plasma 1,25(OH)(2)D. However, the detailed mechanism of thyroid hormone action on vitamin D metabolism is still poorly understood. The present study determined whether renal CYP27B1 gene expression was negatively regulated by thyroid hormones. T(3)-induced hyperthyroid mice showed marked decreases in plasma 1,25(OH)(2)D levels and in renal expression of CYP27B1 mRNA but no changes in plasma concentrations of calcium, PTH, or fibroblast growth factor-23. In addition, we observed that T(3) administration significantly decreased plasma 1,25(OH)(2)D and renal CYP27B1 mRNA levels that were increased by low-calcium or low-phosphorus diets and induced hypocalcemia in mice fed a low-calcium diet. Promoter analysis revealed that T(3) decreases the basal transcriptional activity of the CYP27B1 gene through thyroid hormone receptors (TRα and TRß1) and the retinoid X receptor α (RXRα) in renal proximal tubular cells. Interestingly, we identified an everted repeat negative thyroid hormone response element (1α-nTRE) overlapping the sterol regulatory element (1α-SRE) and the TATA-box -50 to -20 base pairs from the human CYP27B1 gene transcription start site. Finally, we established that CYP27B1 gene transcription is positively regulated by SRE-binding proteins and that a T(3)-bound TRß1/RXRα heterodimer inhibits SRE-binding protein-1c-induced transcriptional activity through the 1α-nTRE. These results suggest that transcriptional repression of the CYP27B1 gene by T(3)-bound TRs/RXRα, acting through the 1α-nTRE, results in decreased renal CYP27B1 expression and plasma 1,25(OH)(2)D levels.

[Lack of phosphorus intake and nutrition].

Since one gram of protein in food provides approximately 15 mg of phosphorus, phosphorus deficiency frequently observed in patients with protein-energy malnutrition (PEM). Chronic phosphorus deficiency in humans causes proximal myopathy. Acute hypophosphatemia may precipitate rhabdomyolysis. Plasma low phosphorus concentration suppresses erythrocyte synthesis and stores of 2,3-diphosphoglycerate (2,3-DPG) , which plays an important role in the affinity of hemoglobin for oxygen. Symptoms of nervous system dysfunction, such as weakness, apathy, a bedridden state, and intention tremors, are also observed in severe hypophosphatemia. Refeeding syndrome is caused by rapid refeeding in PEM, characterized by hypophosphatemia and has metabolic and clinical complications. This is potentially fatal, yet is preventable. Awareness and identification of at-risk patients is crucial to improving management.

Up-regulation of stanniocalcin 1 expression by 1,25-dihydroxy vitamin D(3) and parathyroid hormone in renal proximal tubular cells.

Stanniocalcin 1 and stanniocalcin 2 are two glycoprotein hormones, which act as calcium phosphate-regulating factor on intestine and kidney. We have previously reported that stanniocalcin 2 expression is positively and negatively controlled by 1,25(OH)(2)D(3) and parathyroid hormone in renal proximal tubular cells. However, it has been unclear whether they regulate the stanniocalcin 1 gene expression. In this study, we identified the opossum stanniocalcin 1 cDNA sequence. The opossum stanniocalcin 1 amino acid sequence had 83% homology with human stanniocalcin 1, and has a conserved putative N-linked glycosylation site. Real-time PCR analysis using opossum kidney proximal tubular (OK-P) cells revealed that the mRNA levels of stanniocalcin 1 gene is up-regulated by both 1,25(OH)(2)D(3) and parathyroid hormone in dose-dependent and time-dependent manners. We also demonstrated that the stanniocalcin 1 expression was increased in parathyroid hormone injected rat kidney. Furthermore, the mRNA expression of stanniocalcin 1 and stanniocalcin 2 were oppositely regulated by phorbol 12,13-myristic acetate, a specific PKC activator. Interestingly, the up-regulation of stanniocalcin 1 gene by 1,25(OH)(2)D(3) and phorbol 12,13-myristic acetate were not prevented in the presence of actinomycin D, an RNA synthesis inhibitor. These results suggest that the stanniocalcin 1 gene expression is up-regulated by 1,25(OH)(2)D(3) and parathyroid hormone through mRNA stabilization in renal proximal tubular cells.

Regulation of renal sodium-dependent phosphate co-transporter genes (Npt2a and Npt2c) by all-trans-retinoic acid and its receptors.

The type II sodium-dependent phosphate co-transporters Npt2a and Npt2c play critical roles in the reabsorption of Pi by renal proximal tubular cells. The vitamin A metabolite ATRA (all-trans-retinoic acid) is important for development, cell proliferation and differentiation, and bone formation. It has been reported that ATRA increases the rate of Pi transport in renal proximal tubular cells. However, the molecular mechanism is still unknown. In the present study, we observed the effects of a VAD (vitamin A-deficient) diet on Pi homoeostasis and the expression of Npt2a and Npt2c genes in rat kidney. There was no change in the plasma levels of Pi, but VAD rats significantly increased renal Pi excretion. Renal brush-border membrane Pi uptake activity and renal Npt2a and Npt2c expressions were significantly decreased in VAD rats. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of human Npt2a and Npt2c genes was increased markedly by ATRA and a RAR (retinoic acid receptor)-specific analogue TTNPB {4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphtalenyl)-1-propenyl] benzoic acid} in renal proximal tubular cells overexpressing RARs and RXRs (retinoid X receptors). Furthermore, we identified RAREs (retinoic acid-response elements) in both gene promoters. Interestingly, the half-site sequences (5'-GGTTCA-3': -563 to -558) of 2c-RARE1 overlapped the vitamin D-responsive element in the human Npt2c gene and were functionally important motifs for transcriptional regulation of human Npt2c by ATRA and 1,25(OH)2D3 (1alpha,25-dihydroxyvitamin D3), in both independent or additive actions. In summary, we conclude that VAD induces hyperphosphaturia through the down-regulation of Npt2a and Npt2c gene expression in the kidney.