High Phosphorus Group Research Articles

Hypomethylation of the LncRNA H19 promoter accelerates osteogenic differentiation of vascular smooth muscle cells by activating the Erk1/2 pathways.

Vascular calcification is a common chronic kidney disease complication. This study aimed to investigate the function of long non-coding RNA (LncRNA) H19 in vascular calcification to explore new therapeutic strategies. We induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) using β-glycerophosphate. Then, we detected the LncRNA H19 promoter methylation status and Erk1/2 pathways using methylation-specific polymerase chain reaction and western blotting, respectively. Compared with the control group, high phosphorus levels induced VSMC calcification, accompanied by increases in LncRNA H19 and the osteogenic marker Runx2 and reduction of the contractile phenotype marker SM22a. LncRNA H19 knockdown inhibited osteogenic differentiation and calcification of VSMCs. However, the suppressed role of VSMC calcification caused by shRNA H19 was partially reversed by simultaneous activation of the Erk1/2 pathways. Mechanically, we found that the methylation rate of CpG islands in the LncRNA H19 promoter region was significantly lower in the high-phosphorus group, and the hypomethylation state elevated LncRNA H19 levels, which in turn regulated phosphorylated Erk1/2 expression. LncRNA H19 promoted osteogenic differentiation and calcification of VSMCs by regulating the Erk1/2 pathways. Additionally, hypomethylation of LncRNA H19 promoter CpG islands upregulated LncRNA H19 levels and subsequently activated Erk1/2 phosphorylation.

A High-Phosphorus Diet Moderately Alters the Lipidome and Transcriptome in the Skeletal Muscle of Adult Mice.

A high phosphorus intake has been associated with various metabolic disorders, including chronic kidney disease, cardiovascular disease, and osteoporosis. Recent studies have demonstrated the effects of dietary phosphorus on lipid and glucose metabolism. This study investigated the impact of a high-phosphorus diet on mouse skeletal muscle lipid composition and gene transcription. Adult male mice (n = 12/group) received either a diet with an adequate (0.3%) or a high (1.2%) phosphorus concentration for 6 weeks. The lipidome analysis showed that among the 17 analyzed lipid classes, the concentrations of three classes were reduced in the high phosphorus group compared to the adequate phosphorus group. These classes were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and lysophosphatidylcholine (LPC) (p < 0.05). Out of the three hundred and twenty-three individual lipid species analyzed, forty-nine showed reduced concentrations, while three showed increased concentrations in the high phosphorus group compared to the adequate phosphorus group. The muscle transcriptome analysis identified 142 up- and 222 down-regulated transcripts in the high phosphorus group compared to the adequate phosphorus group. Gene set enrichment analysis identified that genes that were up-regulated in the high phosphorus group were linked to the gene ontology terms "mitochondria" and "Notch signaling pathway", whereas genes that were down-regulated were linked to the "PI3K-AKT pathway". Overall, the effects of the high-phosphorus diet on the muscle lipidome and transcriptome were relatively modest, but consistently indicated an impact on lipid metabolism.

Acute High Dietary Phosphorus Following Low-Phosphorus Diet Acclimation Does Not Enhance Intestinal Fractional Phosphorus Absorption in Nephrectomized Male Rats.

Dietary phosphorus restriction and phosphorus binders are commonly prescribed for patients with chronic kidney disease (CKD). However, occurrences of non-adherence to these interventions are common. As low-phosphorus (LP) diets have been consistently experimentally shown in vitro to increase intestinal phosphorus absorption efficiency, a bout of non-adherence to diet or binders may cause an unintended consequence of enhanced intestinal phosphorus absorption. Thus, we aimed to determine the effect of a single bout of high-phosphorus (HP) intake after acclimation to a LP diet. Male Sprague Dawley rats with 5/6 nephrectomy (n=36) or sham operation (n=36) were block-randomized to 1 of 3 diets: LP (0.1% P w/w), HP (1.2%), or LP followed by acute HP (LPHP 0.1% then 1.2%). Phosphorus absorption tests were conducted using 33P radioisotope administrated by oral gavage or intravenously (iv). Although the overall two-way ANCOVA model for intestinal fractional phosphorus absorption was non-significant, exploratory comparisons showed intestinal fractional phosphorus absorption efficiency tended to be higher in rats in the LP compared with HP or LPHP groups. Rats in the HP or LPHP groups had higher plasma phosphorus compared with rats in the LP group, but the LPHP group was not different from the HP group. Gene expression of the major intestinal phosphate transporter, NaPi-2b, was lower in the jejunum of rats in the LPHP group compared with rats in the HP group but not different in the duodenum. These results demonstrate that an acute HP load after acclimation to a LP diet does not lead to enhanced intestinal fractional phosphorus absorption efficiency in 5/6 nephrectomized male rats. These data provide evidence against the notion that dietary phosphorus restriction or binder use adversely increases absorption efficiency after a single instance of dietary or binder non-adherence. However, other adverse consequences of fluctuating dietary phosphorus intake cannot be ruled out. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

MO548: Endothelial Cell Derived Exosomes Promote Vascular Smooth Muscle Cell Calcification and Senescence by Regulating the Β-Catenin Signaling Pathway Through miRNA-29B-3p

Abstract BACKGROUND AND AIMS Vascular calcification is an important risk factor for high incidence of cardiovascular events and death in patients with chronic kidney disease (CKD). The mechanism of how the calcification signal in early CKD is transmitted to vascular smooth muscle cells (VSMCs) from endothelial cells in the tunica intima to initiate the whole process of vascular calcification remains to be elucidated. The aim of this study was to investigate the effect of miRNA-29B-3p in exosomes derived from vascular endothelial cells under high phosphorus stimulation on VSMC calcification and senescence. METHOD Firstly, human umbilical vein endothelial cells (HUVECs) were divided into the high-phosphorus group (HP-HUVEC, 3mmol/L) and the control group (NP-HUVEC) for exosomes isolation by superspeed centrifugation. Transmission electron microscopy, molecular size analysis and Western blot analysis of exosome markers were used to identify exosomes. Secondly, VSMCs were treated with HP-HUVEC-Exo, NP-HUVEC-Exo and blank control. Then, HUVECs were transfection with miR-29b-3p mimic, miR-29b-3p inhibitor and their controls for the extraction of exosomes. VSMCs were further treated with Exo-miR-29b-3p mimic, Exo-miR-29b-3p inhibitor and their controls. Western Blot was used to detect the expressions of calcification-related protein Runx2, senescence-related protein P21 and β-catenin pathway-related proteins (GSK-3β and β-catenin). Alizarin red staining, ALP activity detection and calcium (Ca) content determination were used to detect the calcification of VSMC. The senescence of VSMC was detected by SA-β-gal staining and the expression of miR-29b-3p was detected by qRT-PCR. RESULTS The expression of marker proteins CD9 and CD63 was observed by Western Blot, the diameter of particle size analysis was about 172.3 ± 72.5 nm and the structure of typical lipid double-layer membrane was observed, suggesting that exosomes were extracted. Compared with the NP-HUVEC-Exo and blank control group, Western Blot showed that the expression of Runx2, P21 and β-catenin was up-regulated and the expression of GSK-3β was down-regulated in VSMC treated with HP-HUVEC-Exo. VSMC treated with HP-HUVEC-Exo also showed up-regulated miR-29b-3p expression, higher ALP activity and Ca content, suggesting exosomes from high phosphorus-stimulated endothelial cells induced calcification and senescence of VSMCs. Compared with the control group, the expression of Runx2 and P21 was up-regulated and showed more significant calcification and aging characteristics and the expression of GSK-3β was down-regulated and the expression of β-catenin was up-regulated in VSMC treated with Exo-miR-29b-3p mimic. VSMC treated with Exo-miR-29b-3p inhibitor showed the opposite results, indicating that miR-29b-3p could repress GSK-3β and activate β-catenin pathway to promoted VSMC calcification/senescence. CONCLUSION Exosomes produced from high phosphorus stimulates endothelial cells could induce the calcification and senescence of VSMC by conveying miR-29b-3p to VSMC and then regulating the β-catenin signaling pathway.

DNMT3A regulates differentiation of osteoblast and autophagy of vascular smooth muscle cells in vascular medial calcification induced by high phosphorus through ERK1/2 signaling

To investigate the effect of DNMT3A in vascular calcification (VC) induced by high phosphorus. The arterial tissues of 12 patients with end stage renal disease (ESRD) and VC and 12 patients with ESRD without VC were collected. Rat vascular smooth muscle cells (VSMCs) were divided into control group, high phosphorus (P) group, P + DMSO group, p-ERK1/2 inhibitor group, DNMT3A group and DNMT3A + P group and P + shRNA-DNMT3A group. Vascular calcification was evaluated by von kossa staining. Cell calcification was evaluated by alizarin red staining. The calcium content was assessed by calcium determination kit. The levels of DNMT3A, Runx2, LC3 and p-ERK1/2 were significantly up-regulated in CKD patients with VC in comparison with those in CKD patients without VC(p<0.05). Moreover, the levels of SM22α and P62 were notably decreased in CKD patients with VC in comparison with those in CKD patients without VC(p<0.05). Similar changes were observed in VSMCs induced by high phosphorus. Knock down of DNMT3A in VSMCs inhibited phenotypic transformation and induced autophagy, then reduced calcification(p<0.05). Moreover, p-ERK1/2 level was downregulated by knock down of DNMT3A in comparison with the control group(p<0.05). In conclusion, DNMT3A regulated high phosphorus induced vascular medial calcification via ERK1/2 signaling.

Severe chronic kidney disease environment reduced calcium-sensing receptor expression in parathyroid glands of adenine-induced rats even without high phosphorus diet

BackgroundChronic kidney disease (CKD) disrupts mineral homeostasis and its main underlying cause is secondary hyperparathyroidism (SHPT). We previously reported that calcium-sensing receptor (CaSR) mRNA and protein expression in parathyroid glands (PTGs) significantly decreased in a CKD rat model induced by a 5/6 nephrectomy that were fed a high phosphorus diet. However, there was a significant difference in the severity of CKD between high phosphorus and adequate phosphorus diet groups. Thus, it was unclear whether CKD environment or the high phosphorus diet influenced CaSR expression, and the underlying mechanism remains largely unknown.MethodsCKD was induced in rats with 0.75% adenine-containing diet. CKD and control rats were maintained for 5 days and 2 weeks on diets with 0.7% or 1.3% phosphorus. For gene expression analysis, quantitative real-time polymerase chain reaction was performed with TaqMan probes. Protein expression was analyzed by immunohistochemistry.ResultsPTG CaSR expression significantly decreased in the presence of a severe CKD environment, even without the high phosphate load. Ki67 expressing cells in PTGs were significantly higher only in the CKD rats fed a high phosphorus diet. Furthermore, among the many genes that could affect CaSR expression, only vitamin D receptor (VDR) and glial cells missing 2 (Gcm2) showed significant changes. Moreover, Gcm2 was significantly reduced at an early stage without significant changes in serum calcium, phosphorus and 1,25(OH)2 vitamin D, and there was no significant reduction in CaSR and VDR expressions. Then, significantly elevated Ki67-positive cell numbers were also only observed in the early CKD PTGs with high-phosphorus diets.ConclusionsOur data suggest that the cause of the decreased PTG CaSR expression is the reduction in VDR and Gcm2 expression; Gcm2 may play a role in the onset and progression of SHPT.

Comparison of the effects of lanthanum carbonate and calcium carbonate on the progression of cardiac valvular calcification after initiation of hemodialysis

BackgroundAlthough mineral metabolism disorder influences cardiac valvular calcification (CVC), few previous studies have examined the effects of non-calcium-containing and calcium-containing phosphate binders on CVC in maintenance hemodialysis patients. The aim of the present study was to compare the effects of lanthanum carbonate (LC) with calcium carbonate (CC) on the progression of CVC in patients who initiated maintenance hemodialysis and to investigate clinical factors related to CVC.MethodsThe current study included 50 subjects (mean age 65 years, 72% males) from our previous randomized controlled trial (LC group, N = 24; CC group, N = 26). CVC was evaluated as CVC score (CVCS) using echocardiography at baseline and 18 months after initiation of hemodialysis. We compared CVCS and the changes between the two groups. We also analyzed the associations between CVCS and any other clinical factors including arterial plaque score (PS) and serum phosphorus levels.ResultsBaseline characteristics of study participants including CVCS were almost comparable between the two groups. At 18 months, there were no significant differences in mineral metabolic markers or CVCS between the two groups, and CVCS were significantly correlated with PS (r = 0.39, p < 0.01). Furthermore, changes in CVCS were significantly correlated with average phosphorus levels (r = 0.36, p < 0.05), which were significantly higher in high serum phosphorus and high PS group compared to low serum phosphorus and low PS group (p < 0.05).ConclusionsIn the present study, there were no significant differences between LC and CC with regard to progression of CVC. However, serum phosphorus levels and arterial plaque seem to be important for the progression and formation of CVC in hemodialysis patients.

Genome-wide identification of lncRNAs and mRNAs differentially expressed in human vascular smooth muscle cells stimulated by high phosphorus

Background Cardiovascular events are the primary cause of death for chronic kidney disease patients, which occurred via vascular calcification evolving pathogenically. Although a high level of phosphorus contributes to the induction of osteogenic differentiation of vascular smooth muscle cells (VSMCs), the role of lncRNA in this process awaits further study. Methods In this study, we systematically investigated the variation of gene expression in human VSMCs induced by high phosphorus. LncRNAs and mRNAs expression were revealed by microarray analyses of the control group and high-phosphorus (HP) group. LncRNA-mRNA co-expression network was established based on the specific lncRNA-mRNA relationships. Hierarchical clustering was used to identify a common set of regulated genes. In addition, Gene Ontology enrichment, Kyoto Gene-Encyclopedia and genomic analyses were conducted for the mRNAs differentially expressed under high phosphorus. Result RT-qPCR results confirmed that the expression of RUNX2, BMP2 and osteocalcin in HP group exhibited significant increases than in control group (p < .05). VSMC in HP group also showed higher intracellular calcium content. Volcano plots results show that 379 mRNAs and 728 lncRNAs different expressed in HP group. LncRNA-mRNA co-expression networks analysis revealed that 8 lncRNAs were the most highly connected lncRNAs. Quantitative analysis indicated that two lncRNAs were confirmed to increase significantly in the HP group. The mRNA expression of NT5E and ICAM1 were higher in group HP, while MAP3K7CL was lower than CON group (p < .05). Conclusion This study provided a working list of lncRNAs that may be relevant to osteogenic differentiation, which presents a new insights into the mechanism of vascular calcification induced by high phosphorus in VSMCs.

Bone marrow mesenchymal stem cell-derived exosomes alleviate high phosphorus-induced vascular smooth muscle cells calcification by modifying microRNA profiles.

Vascular calcification is a common complication in patients with chronic kidney disease (CKD). It is an important predictor of cardiovascular disease and all-cause mortality. Previous studies have confirmed that bone marrow mesenchymal stem cell (BMSC) therapy can reduce vascular calcification, but the specific mechanism is still controversial. In this study, we aimed to investigate the mechanisms of BMSC-derived exosomes (EXO) in improving vascular calcification. BMSCs were cultured and EXO were isolated using the Total Exosome Isolation Reagent. Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured into three groups: control group, high phosphorus group, and high phosphorus plus EXO group. Then, indicators related to smooth muscle cell calcification and microRNA profiles were analyzed. BMSC-derived exosomes inhibited high phosphorus-induced calcification in HA-VSMCs. Besides, EXO treatment reduced calcium content and decreased the alkaline phosphatase (AKP) activity in high phosphorus co-incubated HA-VSMCs. MicroRNA (miRNA) and mRNA expression profiles analyses revealed that 63 miRNAs were significantly upregulated and 1424 genes were significantly downregulated in HA-VSMCs after EXO treatment. Functional miRNA-gene regulatory network revealed that mTOR, MAPK, and Wnt signaling pathway were involved in vascular calcification. BMSC-derived exosomes alleviated high phosphorus-induced calcification in HA-VSMC through modifying miRNA profiles.

The expression of Akt/mTOR in VSMC calcification induced by high phosphate and its regulation of Cbfα1

Objective: To observe the expression of protein kinase B (Akt) / mammalian target of rapamycin (mTOR) induced by high phosphorus in rat vascular smooth muscle cells (VSMC) calcification model, and its modulation on the expression of core binding factor alpha 1 (Cbfα1). Methods: Rat VSMC cells were cultured in vitro, and then divided into two groups: normal phosphorus group (Pi 1.3 mmol/L) and high phosphorus group (Pi 2.6 mmol/L). At day 7, calcium deposition was detected by Alizarin stain. The mRNA levels of Cbfα1 and osteopontin (OPN) were determined by real-time PCR. The protein expressions of p-Akt (ser473), p-mTOR (S2448), Cbfα1 and OPN were quantified by Western blot. Then, VSMC cultured with high phosphorus were treated with Akt inhibitor (Wortmannin) and mTOR inhibitor (Rapamycin) with different concentrations. After 24 h, the mRNA levels of Cbfα1 and OPN were determined and after 48 h, the protein expressions of p-Akt, p-mTOR, Cbfα1 and OPN were quantified. Also, at day 7, calcium deposition was also visualized by Alizarin stain. Results: After 7 days, compared with normal phosphorus group, calcium deposition was more obvious in high phosphorus group. The mRNA expressions of Cbfα1 and OPN increased significantly and the protein expressions of p-Akt, p-mTOR, Cbfα1 and OPN up-regulated significantly in high phosphorus group (all P<0.05). After treated with Wortmannin or Rapamycin for 24 h, compared with high phosphorus group, the mRNA expressions of Cbfα1 and OPN decreased significantly in high phosphorus + Wortmannin (30, 50 and 100 nmol/L) groups (all P<0.05) and high phosphorus + Rapamycin (1, 10 and 100 ng/ml) groups (all P<0.05). After treated with Wortmannin or Rapamycin for 48 h, compared with high phosphorus group, the protein expressions of p-Akt, Cbfα1 and OPN down-regulated significantly in high phosphorus + Wortmannin (30, 50 and 100 nmol/L) groups (all P<0.05). It showed a dose-dependent down-regulation of p-mTOR, Cbfα1 and OPN in high phosphorus + Rapamycin (1, 10, 100 ng/ml) groups (all P<0.05). After 7 days, compared with high phosphorus group, calcium deposition decresased significantly in high phosphorus + Wortmannin and high phosphorus + Rapamycin groups. Conclusions: High phosphorus can induce VSMC calcification. Akt and mTOR are involved in VSMC calcification induced by high phosphorus through the activation of Cbfα1.

Retracted: Activation of the Nrf2‐ARE Signaling Pathway Prevents Hyperphosphatemia‐Induced Vascular Calcification by Inducing Autophagy in Renal Vascular Smooth Muscle Cells

This study investigates the effect of nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) signaling pathway in vascular calcification (VC) via inducing Autophagy in renal vascular smooth muscle cells (VSMCs). VSMCs were assigned into six experimental groups: the normal control, high phosphorus, si-negative control (si-NC), Nrf2-siRNA, over-expressed Nrf2, and negative control (NC) groups. RT-PCR was applied to detect the mRNA expressions of the desired Nrf2-ARE signaling pathway-related genes (Nrf2, NQO-1, HO-1, γ-GCS). The protein products of these genes: apoptosis-related genes (LC3I and LC3II), osteogenic marker proetins (Runt-related transcription factor 2) Runx2 and BMP2 were all detected by Western blotting. Autophagosomes in VSMCs were observed under a transmission electron microscope. We discovered an increased calcium ion concentration and upregulated Runx2, BMP2, Nrf2, HO-1, γ-GCS, NQO-1, and LC3II/LC3I expressions in the high phosphorous, si-NC and Nrf2-siRNA, and NC groups, compared with the normal control group. Compared to the high phosphorus and si-NC groups, higher levels of Runx2 and BMP2 but decreased Nrf2, HO-1, γ-GCS, NQO-1, and LC3II/LC3I expressions were detected in the Nrf2-siRNA group. The high phosphorus, si-NC and over-expressed Nrf2 experimental groups all had increased Nrf2, NQO-1, HO-1, γ-GCS, and LC3II/LC3I expressions as well as high numbers of autophagosomes compared with the normal control group. Finally, we detected a lower amount of autophagosomes presence and Nrf2, NQO-1, HO-1 γ-GCS, and LC3II/LC3 protein expression of Nrf2-siRNA group than that of the high phosphorus and si-NC groups. Activation of Nrf2-ARE signaling pathway may prevent hyperphosphatemia-induced VC by inducing autophagy in VSMCs. J. Cell. Biochem. 118: 4708-4715, 2017. © 2017 Wiley Periodicals, Inc.

Effect of a high phosphorus diet on indicators of renal health in cats.

Objectives High phosphorus intake may further impair renal health in cats with chronic kidney disease (CKD). The hypothesis that a high phosphorus (HP) diet might be nephrotoxic for healthy animals was tested in cats, a species with a high incidence of naturally occurring CKD. Methods Thirteen healthy adult cats were fed a phosphorus excess diet (about five times maintenance requirements), and this HP group was compared with cats on a balanced control diet (CON). The trial lasted for 29 days (10 days of faeces and urine collection). Endogenous creatinine clearance was determined towards the end of the trial. Fresh urine was tested for glucose and proteins. Results Glucosuria and microalbuminuria were observed exclusively in the HP group in 9/13 cats. Creatinine clearance was significantly decreased after feeding HP. In the HP group phosphorus was highly available (apparent digestibility around 60%). Renal phosphorus excretion was significantly increased in the HP group (115 mg/kg body weight/d vs 16 mg/kg body weight/d in the CON group). Conclusions and relevance The intake of a diet with an excessive content of highly available phosphorus may have adverse effects on parameters of kidney function in healthy cats.

Effects of intermediate conductance calcium-activated potassium channel blocker TARAM-34 on β-glycerophosphate induced vascular smooth muscle cells calcification

To observe the role of TRAM-34 (1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole), the blocker of intermediate conductance calcium-activated potassium channel (KCa3.1), on β-glycerophosphate induced vascular calcification in vitro. Vascular smooth muscle cells(VSMCs) were obtained from rat thoracic aorta, and VSMCs after the fourth passage and aortic rings were divided into control group (cultured in DMEM with 10% fetal bovine serum), high phosphorus group (cultured in DMEM with 10% fetal bovine serum and 10% β-glycerophosphate) and TRAM-34 group(20 nmol/L TRAM-34 was added into high phosphorus DMEM). Calcium deposition of VSMCs and aortic rings were measured by o-cresolphthalein complexone method.Calcium influx of VSMCs was measured by immunofluorescence probe Fluo-3 AM.The expression of runt-related transcription factor 2(Runx2)was detected by RT-PCR and Western blot for cells and immunohistochemistry for aortic rings.ALP activity was measured by alkaline phosphatase activity detection kit. (1) Compared with control group, calcification was significantly increased in high phosphorus group ((121.67±6.17) mg/g vs. (84.38±8.17) mg/g, P<0.05) and this effect could be attenuated by TRAM-34 ((93.31±11.36) mg/g, P<0.05 vs. high phosphorus group) after 12 days culture. Similar results were found in aortic rings cultured for 12 days-high phosphorus group: (7.17±0.57) mg/g vs. (1.18±0.13) mg/g (P<0.05) and TRAM-34: (4.71±0.42) mg/g, P<0.05 vs. high phosphorus group.(2) Compared with control group, the calcium influx was higher in high phosphorus group (349.22±40.47 vs. 151.67±16.94, P<0.05) and reduced in TRAM-34 group (194.67±22.21, P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days. (3) Both mRNA and protein expressions of Runx2 in high phosphorus groups were higher than in control group (0.630±0.033 vs.0.340±0.058 and 0.865±0.031 vs.0.414±0.011, both P<0.05) and lower in TRAM-34 group (0.399±0.023 and 0.575±0.014, both P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days.Besides, compared with high phosphorus group, the expression of Runx2 was decreased in control group(0.113±0.010 vs.0.067±0.008, P<0.05) and TRAM-34 group (0.069±0.006, P<0.05) after aortic rings were cultured for 4 days. (4) Compared with control group, the activity of ALP was significantly increased in high phosphorus group (96.56±9.84 vs.46.92±4.60, P<0.05) and decreased in TRAM-34 group(70.20±8.41, P<0.05 vs. high phosphorus group) in VSMCs simulated for 12 days. KCa3.1 blocker TRAM-34 can inhibit β-glycerophosphate induced VSMCs and aortic ring calcification through inhibiting calcium influx, downregulating Runx2 expression and attenuating osteogenic differentiation.

Effect of hyperphosphatemia on gene expression of the Na-Pi cotransporter in rats.

We investigated the effect of high phosphorus content on the sodium-phosphate cotransporter (NaPi-IIa and NaPi-IIl). Forty-eight Sprague-Dawley rats were divided into 3 groups: high-phosphorus group (HP) with fructose diphosphate sodium injection; self-manufactured low-phosphorus diet group (LP); and normal diet group (NP). At the 1st, 2nd, 4th, and 6th weeks, 4 rats from each group were sacrificed for detecting serum levels of calcium, phosphorus, and intact parathyroid hormone. Semi-quantitative retrovirus-polymerase chain reaction was used to detect the expression of NaPi-IIa and NaPi-III mRNA in kidney. At the 1st, 2nd, 4th, and 6th weeks, serum phosphorus and parathyroid hormone levels in HP group were significantly higher than those in LP and NP groups (P < 0.05). Serum calcium levels in the 3 groups showed no difference (P > 0.05). Comparing the expression of NaPi-IIa mRNA in HP group with LP and NP groups, NaPi-IIa mRNA expression was significantly reduced in HP group (P < 0.05), while NaPi-IIa mRNA expression in LP group began increasing at the 4th week (P < 0.05). At the 1st, 2nd, and 4th weeks, the expression of NaPi-III mRNA in HP, LP, and NP groups showed no clear differences (P > 0.05), while at the 6th week in HP group, NaPi-III mRNA expression was slightly increased compared to in LP and NP groups (P < 0.05). Hyperphosphatemia significantly affected NaPi-IIa and NaPi-III mRNA expression, and a factor promote an increase in intact parathyroid hormone independently of calcium.

Effects of Excess Dietary Phosphorus on Fecal Phosphorus Excretion and Water Extractable Phosphorus in Horses

Phosphorus excretion was studied in horses fed excess phosphorus. The hypothesis of this study was that the concentration of phosphorus and water extractable phosphorus (WEP) in the feces of sedentary horses would reflect the level of dietary phosphorus. Eight Standardbred mares were divided into two groups and received diets of grass hay and grain. The high phosphorus (HP) group received 142 g/d of monosodium phosphate (NaH2PO4), formulated to provide 4.5-times dietary phosphorus requirement, or 65-g phosphorus per day. The low phosphorus (LP) group received 28 g of phosphorus per day in the basal diet. These amounts were based on horses consuming 2% of body weight per day as hay plus supplemental grain. After a 7-day diet adaptation, a 5-day collection was conducted. Animals were housed from 4 PM until 8 AM the following morning without bedding during the collection period. After the first period, horses underwent a 10-day washout and then groups were crossed over for a second 7-day adaptation and 5-day collection. Feces was collected daily, weighed, and a 10% of aliquot taken. At the end of each collection period, feces was composited for each horse and analyzed for nitrogen, potassium, phosphorus, and WEP. Fecal phosphorus and WEP content were greater in the HP group (8.1 ± 0.3 vs. 6.8 ± 0.3 g/kg, respectively) than the LP group (3.6 ± 0.3 vs. 2.1 ± 0.3 g/kg, respectively; P < .05). Overfeeding a phosphorus supplement increased phosphorus and WEP in the manure; WEP may be useful for determining phosphorus runoff risk.

High Phosphorus Diet-Induced Changes in NaPi-IIb Phosphate Transporter Expression in the Rat Kidney: DNA Microarray Analysis

The mechanism by which phosphorus levels are maintained in the body was investigated by analyzing changes in gene expression in the rat kidney following administration of a high phosphorus (HP) diet. Male Wistar rats were divided into two groups and fed a diet containing 0.3% (control) or 1.2% (HP) phosphorous for 24 days. Phosphorous retention was not significantly increased in HP rats, but fractional excretion of phosphorus was significantly increased in the HP group compared to controls, with an excessive amount of the ingested phosphorus being passed through the body. DNA microarray analysis of kidney tissue from both groups revealed changes in gene expression profile induced by a HP diet. Among the genes that were upregulated, Gene Ontology (GO) terms related to ossification, collagen fibril organization, and inflammation and immune response were significantly enriched. In particular, there was significant upregulation of type IIb sodium-dependent phosphate transporter (NaPi-IIb) in the HP rat kidney compared to control rats. This upregulation was confirmed by in situ hybridization. Distinct signals for NaPi-IIb in both the cortex and medulla of the kidney were apparent in the HP group, while the corresponding signals were much weaker in the control group. Immunohistochemical analysis showed that NaPi-IIb localized to the basolateral side of kidney epithelial cells surrounding the urinary duct in HP rats but not in control animals. These data suggest that NaPi-IIb is upregulated in the kidney in response to the active excretion of phosphate in HP diet-fed rats.

Analysis of the level of Serum 1,25-Dihydroxy Cholecalciferol in 30 children with Endogenous Hyperphosphatemia

Objective To explore the effect of the level of 1,25-Dihydroxy Cholecalciferol in 30 cases of children with endogenous hyperphosphatemia. Methods Using fasting blood and OLPUMS AU400, determination of phosphorus by Phosphomolybdate ultraviolet France, determination of serum calcium by methyl thymol blue spectrophotometry (MTB) colorimetric, Determination of 1,25 (OH)2D3 levels in 30 cases of high-phosphorus blood group and 30 cases of normal serum phosphate group by Ydioimmunoassay. Results The phosphorus and calcium phosphate product in hyperphosphatemia group is significantly higher than the normal serum phosphate group. The difference is statistically significant. The calcium is slightly lower than the normal serum phosphate in hyperphosphatemia group, The difference is not significant ( P > 0. 05 ). The 1,25 (OH)2D3 of hyperphosphatemia group is significantly lower than the normal serum phosphate group. The difference ia statistically significant( P < 0. 01 ). Conclusion Hyperphosphatemia can inhibit the synthesis of 1,25 (OH)2D3. Key words: Hyperphosphatemia; 1,25 Dihydroxy Cholecalciferol

Effect of ammonium chloride and dietary phosphorus in the azotaemic rat. Part II--Kidney hypertrophy and calcium deposition.

Kidney hypertrophy is stimulated by both partial nephrectomy and NH(4)Cl administration. Also, parathyroidectomy (PTX) has been reported to prevent kidney hypertrophy induced by a high protein diet. Our goal was to determine in the azotaemic rat: (i) the combined effects of NH(4)Cl administration and dietary phosphorus on the development of kidney hypertrophy and calcium deposition in the kidney and (ii) whether the absence of parathyroid hormone (PTH) affected the development of kidney hypertrophy and calcium deposition. High (HPD, 1.2%), normal (NPD, 0.6%) or low (LPD, <0.05%) phosphorus diets were given to 5/6 nephrectomized rats for 30 days. In each dietary group, one-half of the rats were given NH(4)Cl in the drinking water. The six groups of rats were: (i) HPD + NH(4)Cl; (ii) HPD; (iii) NPD + NH(4)Cl; (iv) NPD; (v) LPD + NH(4)Cl and (vi) LPD. In a separate study, PTX was performed to determine whether PTH affected renal hypertrophy in 5/6 nephrectomized rats given NH(4)Cl. Both with and without NH(4)Cl (+/-NH(4)Cl), kidney weight was greatest (P<0.05) in the HPD groups. In each dietary phosphorus group, kidney weight was greater (P<0.05) in the NH(4)Cl group. In both the +/-NH(4)Cl groups, kidney calcium content was greatest (P<0.05) in the HPD group, but was less (P<0.05) in the NPD and HPD groups given NH(4)Cl. An inverse correlation was present between creatinine clearance and kidney calcium content (r = -0.51, P<0.001). When factored for kidney weight, creatinine clearance was less (P<0.05) in the HPD group in both the +/-NH(4)Cl groups, but was greater in the HPD + NH(4)Cl than in the HPD group. In PTX rats, kidney weight was greater (P<0.05) and kidney calcium deposition was less (P<0.05) in rats given NH(4)Cl. In azotaemic rats studied for 30 days, NH(4)Cl administration induced kidney hypertrophy. A HPD also induced kidney hypertrophy. The effects on kidney calcium deposition were divergent for which NH(4)Cl administration decreased and a HPD increased calcium deposition. The inverse correlation between kidney calcium content and creatinine clearance suggests that kidney calcium deposition is harmful to renal function. When factored for kidney weight, the lower creatinine clearance in the high phosphorus group suggests that kidney hypertrophy does not completely compensate for the harmful effects of a HPD. This result also suggests that a longer study would probably result in more rapid deterioration in the high phosphorus group. In PTX rats, the absence of PTH did not prevent NH(4)Cl from inducing kidney hypertrophy and reducing kidney calcium deposition. In conclusion, NH(4)Cl and dietary phosphorus each independently affect kidney growth and calcium deposition in the growing rat with renal failure.

Effect of phosphorus supply on mineral balance at high calcium intakes in very low birth weight infants

To evaluate the effect of varying phosphorus intake with constant high calcium intake (430 micrograms/kJ, or 180 mg/100 kcal), we randomly assigned 35 appropriate-for-gestational-age healthy male infants (birth weight, 715-1510 g) on day 21 postnatally to either standard-phosphorus (215 micrograms/kJ, or 90 mg/100 kcal), moderate-phosphorus (254 micrograms/kJ, or 106 mg/100 kcal), or high-phosphorus intake (287 micrograms/kJ, or 120 mg/100 kcal). Three-day mineral balances were determined after 7 d of the study diets. Weight and head circumference gain and intake of energy and vitamin D were not different for all groups. Calcium retentions were sufficient to meet intrauterine accretion in all groups. Phosphorus calculated to be available for soft tissue was significantly higher in the moderate- and high-phosphorus groups, and was sufficient to support soft tissue phosphorus accretion in these two groups. Total absorbed phosphorus and phosphorus tubular reabsorption were each affected by phosphorus intake. We conclude that very low birth weight infants fed high calcium may require greater phosphorus intake than that provided by formulas containing a ratio of calcium to phosphorus of 2:1. We suggest that the optimal mass ratio of calcium to phosphorus for formula for very low birth weight infants is from 1.6:1 to 1.8:1.

Mechanical properties of metacarpal bones of foals fed different energy and phosphorous levels

SummaryThe mechanical and geometric properties of metacarpal bones of 41 Quarter Horse yearlings fed diets with different energy and phosphorus levels were tested and compared. A 2 times 3 factorial experiment was used to compare 2 energy sources and 3 levels of dietary phosphorus. The 2 energy sources were based either on alfalfa (2.65 Mcal digestible energy [DE]/kg dry matter [DM] feed) or on grain (3.09 Mcal DE/kg DM). In each diet type, the 3 phosphorus concentrations included: an unsupplemented, low phosphorus (LP) diet; a normal phosphorus (NP) diet supplemented to meet NRC (1978) requirements for weanling foals; and a high phosphorus (HP) diet supplemented to supply 150% of 1978 NRC phosphorus requirements. At the completion of the feeding trial, the yearling horses were destroyed and the metacarpal bones recovered. The left metacarpus was serially sectioned at 0.5 cm thicknesses with a band saw and a section was sampled for histology and measurement of geometric properties including the rectangular moments of inertia (Ix, Iy), the products of inertia and principal moments of inertia (Ixx, Iyy). The moments of inertia were then recalculated to the change in orientation of the x‐y axes and symbolised Ix1 and Iy1. Energy intakes were similar despite differences in energy content of the forage and grain diets. However, forage‐fed yearlings had lower (p&lt;0.0589) breaking strengths (23.4 kilograms per square inch [ksi] than grain‐fed horses (25.4 ksi). The geometric properties between LP and NP groups did not differ. However, the geometric properties (Ix1, Ixx, Iyy) differed (p&lt;0.05) between the HP groups and the LP and NP groups. Within the NP group, (Iyy) differed (p&lt;0.038) between males and females. The right metacarpus, with both small metacarpal bones removed but with the periosteum intact, was loaded in a 4‐point bending until failure. The metacarpal bones were placed in a specially‐made Instron tester made so that the mid‐diaphyses were loaded. All bones failed in a single plane at right angles to the main axis of the bone. Seventeen of the 41 bones (41.5%) fractured through the nutrient foramen. The mechanical properties of males and females did not differ except in the NP group wherein stiffness of the metacarpal bone of females was lower (p&lt;0.05). In the NP group, males were 14% heavier (p&lt;0.0384) than females. Although most mechanical properties did not differ between the LP and NP groups, the HP group had a higher load at yield point (6397 N) than the LP group (5789 N). Phosphorus intakes, therefore, affect both mechanical strength and geometric properties of growing horses.