high phosphorus ( p ) intake is known as one of the risk factors for impaired bone health .
several researchers have investigated the adverse effects of a high p diet on bone metabolism . in human adults ,
a diet containing p additives increases urinary hydroxyproline excretion , a bone resorption marker . in growing male rats ,
elevation in parathyroid hormone ( pth ) secretion is considered one of the mechanisms by which a high p diet impairs bone metabolism .
pth stimulates osteoblasts , which produce mediators of osteoclastic bone resorption such as macrophage colony - stimulating factor ( m - csf ) , interleukin-6 ( il-6 ) , or receptor activator of nf-b ligand ( rankl ) [ 46 ] .
we previously reported that a high p diet increased rankl mrna expression and the osteoclast number in rats .
it could therefore be deduced that high p diet - induced elevated pth secretion leads to an increase in rankl expression , which enhances osteoclastic bone resorption .
overton and basu suggested that bone loss occurs with increasing age at a rate of approximately 1% per year averaged over the age range of 2976  y . in a previous mouse study by ferguson et al . , bone mass and mechanical properties approached mature levels by 12 weeks of age while age - related osteopenia was observed after 42 weeks of age .
we previously reported the effects of a high p diet on mechanical properties of the femur in 4- , 12- , 24- , and 80-week - old mice .
the results showed that a high p diet decreased the breaking force of the femur in 80-week - old mice and the stiffness of the femur in 24- and 80-week - old mice .
we also found that a high p diet increased serum pth concentration in 12- , 24- , and 80-week - old mice , and 80-week - old mice had a higher serum pth concentration than mice at other ages .
therefore , it was thought that a high p diet strongly influences aged mice in terms of pth response .
the purpose of this study was to clarify the mechanism by which a high p diet affects bone metabolism in older mice .
we assessed the changes in bone metabolism in young and aged mice fed a high p diet by measuring the mrna expression of bone metabolism mediators using real - time polymerase chain reaction ( pcr ) .
the mice were maintained in accordance with the university guidelines for the care and use of laboratory animals .
the experimental diets were based on the ain-93 g diet ( table 1 ) . a control diet containing 0.3% p and a high p diet containing 1.2%
twenty - four 10-week - old ddy male mice were purchased from slc ( shizuoka , japan ) and housed individually in stainless cages in a room maintained at 22c with a 12-hour light / dark cycle .
half of the mice were fed a commercial diet ( ce-2 , clea japan , tokyo , japan ) until 78 weeks of age .
after the acclimatization period , 12 young ( 12-week - old ) and 12 aged ( 80-week - old ) mice were randomly divided into two experimental groups and fed the control diet or the high p diet for 4 weeks . they were given free access to the diets and distilled water .
their urine samples were collected during the 5 days prior to euthanasia for the further analyses . at the end of the experimental period
, all mice were euthanized under anesthesia and blood , bone , and duodenum samples were collected for analyses .
the serum ca level was analyzed by atomic absorption spectrophotometry ( hitachi a-2000 ; hitachi , tokyo , japan ) according to the method of gimblet et al . .
the serum p level was analyzed by colorimetry using phospha c - test wako ( wako pure chemical industries , osaka , japan ) .
serum pth concentration was measured using the mouse intact pth enzyme - linked immunosorbent assay ( elisa ) kit ( alpco , nh , usa ) .
serum intact osteocalcin ( oc ) concentration was measured using the mouse osteocalcin eia kit ( biomedical technologies , ma , usa ) .
urinary c - terminal telopeptide of type i collagen ( ctx ) level was measured using ratlaps eia kit ( immunodiagnostic systems , boldon , uk ) .
urinary creatinine level was measured using the jaffe reaction , as described by lustgarten and wenk .
total rna was isolated from the homogenized femurs or duodenum by using trizol reagent ( life technologies , ca , usa ) according to the manufacturer 's specifications .
the amount and purity of the rna were assessed using a nanodrop 2000c ( thermo fisher scientific , ma , usa ) .
complementary dna ( cdna ) was synthesized using the high - capacity rna - to - cdna kit ( applied biosystems , ca , usa ) . for real - time pcr
, the reaction mixture was prepared using the taqman gene expression master mix ( applied biosystems ) with taqman gene expression assays ( applied biosystems ) for mouse pth receptor ( assay i d : mm00441046_m1 ) , mouse rankl ( assay i d : mm00441906_m1 ) , mouse osteoprotegerin ( opg ) ( assay i d : mm01205928_m1 ) , mouse tartrate resistant acid phosphatase ( trap ) ( assay i d : mm00475698_m1 ) , mouse runt related transcription factor 2 ( runx2 ) ( assay i d : mm00501580_m1 ) , mouse osterix ( assay i d : mm04209856_m1 ) , mouse alkaline phosphatase ( alp ) ( assay i d : mm00475834_m1 ) , mouse osteopontin ( opn ) ( assay i d : mm00436767_m1 ) , mouse oc ( assay i d : mm03413826_mh ) , mouse type i collagen ( col1a1 ) ( assay i d : mm00801666_g1 ) , mouse transient receptor potential vanilloid type 6 ( trpv6 ) ( assay i d : mm00499069_m1 ) , mouse calbindin - d9k ( assay i d : mm00486654_m1 ) , mouse plasma membrane ca - atpase 1b ( pmca1b ) ( assay i d : mm01245805_m1 ) , and mouse glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) ( assay i d : mm99999915_g1 ) .
real - time pcr was performed using a stepone real - time pcr system ( applied biosystems ) .
the value of the young mice fed the control diet was considered to be 1.00 .
after two - way analysis of variance ( anova ) , fisher 's protected least significant difference ( plsd ) test was used to determine significant differences among the groups .
in both young and aged mice , there was no significant difference in the initial body weight between mice fed the control and high p diets ( table 2 ) .
the initial body weight of aged mice was significantly higher than that of young mice .
there were no significant differences in serum ca and p concentrations among the groups ( table 2 ) . in both young and aged mice ,
although there was no significant difference in serum pth concentration between young and aged mice fed the control diet , serum pth concentration was significantly higher in aged mice than in young mice fed the high p diet . in both young and aged mice ,
the high p diet significantly increased serum oc concentration ( table 2 ) . in mice fed both
the control and high p diets , serum oc concentration was significantly lower in aged mice than in young mice . in both young and aged mice ,
although there was no significant difference in urinary excretion of ctx between young and aged mice fed the control diet , urinary excretion of ctx was significantly lower in aged mice than in young mice fed the high p diet . in both young and aged mice , the high p diet significantly increased mrna expression of pth receptor , rankl , trap , runx2 , osterix , alp , opn , oc , and col1a1 ( figure 1 ) compared to the control diet . in mice
fed the control and high p diets , mrna expression of pth receptor , rankl , trap , runx2 , osterix , alp , opn , oc , and col1a1 was significantly lower in aged mice than in young mice .
the high p diet significantly increased rankl / opg ratio in aged mice but did not in young mice . in both young and aged mice ,
high p diet significantly increased mrna expression of trpv6 , cabp9k , and pmca1b ( figure 2 ) compared to the control diet . in mice
fed the control and high p diets , mrna expression of trpv6 and cabp9k was significantly lower in aged mice than in young mice .
in humans , bone loss occurs with increasing age at a rate of approximately 1% per year averaged over the ages of 2976 years .
therefore , aging is one of the risk factors for bone loss . in a previous mouse study ,
bone mass and mechanical properties were shown to approach mature levels by 12 weeks of age , and age - related osteopenia was observed after 42 weeks . in this study , we investigated bone metabolism by measuring markers of bone formation and resorption , serum oc concentration , and urinary ctx excretion .
serum oc concentration was significantly lower in aged mice than in young mice fed the control diet , whereas there is no difference in urinary excretion of ctx between young and aged mice fed the control diet .
these results showed that aged mice present a decrease in bone formation , and it appears that the balance between bone formation and resorption may be disrupted in aged mice .
bone formation is mediated by osteoblasts . using gene - deficient mouse models , runx2 and osterix
were shown to be essential transcription factors for osteoblast differentiation and bone formation [ 15 , 16 ] . in this study ,
runx2 and osterix mrna expression were significantly lower in aged mice than in young mice .
these results suggest that aging leads to a reduction in osteoblast differentiation and that runx2 and osterix mrna expression changes are associated with a decrease in bone formation in aged mice .
consequently , decreases in mrna expression of alp and bone matrix proteins such as opn , oc , and col1a1 also occurred in aged mice .
ikeda et al . showed that the mrna expression of opn , oc , and col1a1 decreased in both cortical and trabecular bones in aged rats compared to young animals .
in addition , cao et al . reported that alp and col1a1 expression declined in aged mice compared to young mice .
thus , aging results in a decrease in bone formation with declined osteoblast - related gene expression . with regard to bone resorption
, this study showed that rankl and trap mrna expression were decreased in aged mice compared to young mice , despite unchanging serum pth concentration .
since pth stimulates rankl , the result of rankl mrna expression seems to contradict that of serum pth concentration .
however , pth receptor mrna expression was decreased in aged mice compared to young mice in this study .
this result suggested that pth action was suppressed , which decreased rankl mrna expression in aged mice .
though urinary excretion of ctx was unchanged , expression of bone resorption - rerated genes was decreased in aged mice compared to young mice .
however , it is generally known that age - related increase in serum pth contributes to the increase in bone resorption .
therefore , results of serum pth concentration and bone resorption between young and aged mice in this study are contradictory .
further studies with the increase in number of mice per group are needed to address these discrepancies .
a decline in intestinal ca absorption is also one of the causes of age - related bone loss .
the transcellular ca pathway , which is affected by 1,25-dihydroxyvitamin d ( 1,25(oh)2d ) , has been proposed to involve ca entry via trpv6 , intracellular diffusion of ca by calbindin - d9k , and basolateral extrusion of ca by pmca1b . in this study ,
trpv6 and calbindin - d9k mrna expression in the duodenum were decreased in aged mice compared to young mice .
demonstrated that plasma 1,25(oh)2d , duodenal calbindin d protein , and ca absorption decreased with age in rats . from this study
therefore , a decrease in serum 1,25(oh)2d might reflect our results on trpv6 and calbindin - d9k mrna expression .
many studies have reported that high p intake induces an increase in serum pth concentration in humans [ 1 , 23 ] and animals [ 2 , 3 , 24 ] . in this study ,
the high p diet increased serum pth concentration in both young and aged mice and was greater in aged mice than in young mice fed the high p diet .
similar to our previous study , these results suggest that the response to a high p diet in terms of pth secretion might be different and greater with aging .
kidney function decreases with age , and declining kidney function causes an increase in pth secretion .
. therefore , the combination of aging and high p diet might be one of the reasons that higher serum pth concentration was observed in aged mice fed the high p diet .
we previously reported that a high p diet increased rankl mrna expression and bone resorption in growing rats .
therefore , it was suggested that elevated pth secretion induced by the high p diet led to an increase in rankl expression , which increased bone resorption . in this study ,
the high p diet significantly increased urinary excretion of ctx in both young and aged mice .
regarding mrna expression of bone resorption - related molecules in the femora , the high p diet significantly increased rankl and trap mrna expressions in both young and aged mice .
rankl actions are inhibited by opg , which acts as a decoy receptor by blocking rankl binding to its receptor . in this study ,
the high p diet significantly increased the rankl / opg ratio in aged mice , whereas the ratio was unchanged in young mice .
many reports have supported the assertion that the increase in rankl / opg ratio promotes osteoclastogenesis , accelerates bone resorption , and induces bone loss .
our previous study showed that a high p diet decreased the breaking force and stiffness of the femur in aged mice compared to young mice .
our findings show that the high p diet in the aged mice leads to increased pth secretion and consequent increases in the rankl / opg ratio accelerating osteoclastogenesis .
previous studies showed that pth regulates runx2 and osterix mrna expression [ 30 , 31 ] . in this study , the high p diet significantly increased serum oc concentration and mrna expression of runx2 , osterix , alp , opn , oc , and col1a1 in both young and aged mice . from the results of bone resorption and formation markers , high bone turnover with resorption exceeding formation
since high bone turnover is a risk factor for bone fracture and osteoporosis , a high p diet might be a risk factor for bone loss not only in young mice but also in aged mice .
pth secretion might reflect a decrease in ca absorption in the high p diet group .
although the mechanism underlying the high p diet - induced decreased ca absorption remains unknown , it is thought that the formation of insoluble ca and p salts in the intestinal lumen is an important factor .
our previous study also showed that high p diet decreased ca absorption in female rats .
while we did not evaluate ca absorption in this study , it is possible to estimate the decrease in ca absorption by high p diet in young and aged mice .
however , this study showed that the high p diet significantly increased mrna expression of trpv6 , cabp9k , and pmca1b in both young and aged mice .
previous study demonstrated that ca restriction during lactation stimulated ca - binding protein and active ca transport in jejunum and ileum .
furthermore , ca deficient diet resulted in an increase in duodenal pmca mrna in chickens .
these studies suggested that 1,25(oh)2d - regulated ca transporters might be stimulated by low ca status in the intestinal lumen .
thus , a decrease in soluble ca induced by high p diet might lead to trpv6 , cabp9k , and pmca1b mrna expression , and these increases in 1,25(oh)2d - regulated gene expression seem to compensate for a decrease in ca absorption by the high p diet . in brief , our results suggest that high p diet accelerates the transcellular ca pathway , though absorbed amount of ca was insufficient to maintain serum pth concentration .
it is known that fibroblast growth factor 23 ( fgf23 ) and 1,25(oh)2d as well as pth are key factors for ca and p metabolism .
therefore , measuring serum fgf23 and 1,25(oh)2d is important to fully elucidate the mechanisms by which high p diet changes ca and p metabolism , and further studies are needed to clarify the details .
in conclusion , we demonstrated that the high p diet increased bone metabolism - related gene expression in both young and aged mice .
furthermore , the high p diet affected pth secretion differently in young and aged mice , leading to an increase in the rankl / opg mrna ratio in aged mice .
 in this study , the effects of high phosphorus ( p ) diet on bone metabolism - related gene expression were investigated in young and aged mice . 
 twelve- and 80-week - old ddy male mice were divided into two groups , respectively , and fed a control diet containing 0.3% p or a high p diet containing 1.2% p. after 4 weeks of treatment , serum parathyroid hormone ( pth ) concentration was significantly higher in the high p groups than in the control groups in both young and aged mice and was significantly higher in aged mice than in young mice fed the high p diet . 
 high p diet significantly increased receptor activator of nf-b ligand ( rankl ) mrna in the femur of both young and aged mice and significantly increased the rankl / osteoprotegerin ( opg ) mrna ratio only in aged mice . 
 high p diet significantly increased mrna expression of transient receptor potential vanilloid type 6 , calbindin - d9k , and plasma membrane ca2 + -atpase 1b in the duodenum of both young and aged mice . 
 these results suggest that high p diet increased rankl mrna expression in the femur and calcium absorption - related gene expression in the duodenum regardless of age . 
 furthermore , the high p diet - induced increase in pth secretion might increase the rankl / opg mrna ratio in aged mice . 
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion