Fibroblast growth factor 23 (FGF23) is associated with cardiovascular disease and all-cause mortality in patients with diabetes mellitus. Insulin resistance has recently been reported to increase FGF23 levels, and resistin is a peptide that mainly regulates insulin resistance. However, few studies have investigated the association between FGF23 and resistin. A total of 422 patients with diabetes mellitus were recruited for this cross-sectional study to examine the association between resistin and intact FGF23. The mean ( ± standard deviation) age was 63.1 ± 11.9 years, and the median HbA1c was 6.7% (range, 6.1-7.1%). The mean estimated glomerular filtration rate (eGFR) was 66.2 ± 23.1 mL/min/m2. Multiple regression analysis for resistin showed that logFGF23 (coefficient (Coef): 1.551; standard error (SE): 0.739; P = 0.036), C-peptide (Coef: 0.798; SE: 0.229; P = 0.001), ghrelin (Coef: 1.061; SE: 0.332; P = 0.001), intact parathyroid hormone (Coef: 0.022; SE: 0.099; P = 0.030), and eGFR (Coef: -0.091; SE: 0.017; P < 0.001) were all significantly associated with the resistin level. These associations were modified in patients with higher age, lower body mass index, and higher vitamin D levels. These results suggest that resistin is positively associated with serum FGF23 levels.

The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

The jejunum plays crucial roles for the digestion and absorption of nutrients and minerals and for barrier functions that are essential for a healthy, productive life cycle of farm animals, including laying hens. Accordingly, knowledge of the molecular pathways that emerge in the intestine during development, and particularly at the beginning of laying activity, will help to derive strategies for improving nutrient efficiency in laying hens. In this study, jejunal samples were obtained from two high-yielding layer strains at five developmental stages (weeks 10, 16, 24, 30 and 60 of life) for RNA-sequencing, alongside the profiling of blood plasma parameters to approximate the dynamics of mineral homeostasis. The results reflected a marked distinction between the pre-laying and laying phase as inferred from levels of parathyroid hormone, triiodothyronine, estradiol, vitamin D, and calcium. Moreover, the expression patterns of the intestinal mucosa responded directly to the changing metabolic and nutritional profiles at the beginning of the laying phase in maturing high-yielding strains of laying hens. These comprise signaling events namely RANK/RANKL signaling and cellular senescence. Taken together, the timing of sexual maturity of laying hens demands closer examination to unravel metabolic requirements and associated endogenous mechanisms.

Nephrolithiasis, secondary hyperparathyroidism (sHPT), and cardiovascular complications are associated with disturbances in Ca handling and contribute to morbidity/mortality during haemodialysis (HD). Calcimimetics, activators of the calcium-sensing receptor (CaSR), provide an effective means of reducing parathyroid hormone (PTH) secretion in sHPT. Polymorphism in CaSR gene (CASR) influences Ca-related parameters, however it was not shown in HD patients for CASR rs7652589. The minor allele at this polymorphism modifies the binding sites of transcription factors and CaSR expression. We hypothesized that CASR rs7652589 variants may also influence CaSR in end stage renal disease (ESRD). We aimed to determine the associations of rs7652589 with nephrolithiasis-related ESRD, Ca, P, ALP, PTH, response to treatment with cinacalcet, prevalence of coronary artery disease, and all-cause/cardiovascular mortality in HD patients (n = 1162). Healthy individuals (n = 918) were controls. This study shows that the A allele of rs7652589 is a risk allele for nephrolithiasis-related ESRD. The AA genotype is associated with more severe sHPT (higher Ca and PTH concentrations). The A allele is associated with reduced CaSR transcript level in peripheral blood mononuclear cells. According to computational analysis, potential binding sites for GLI3, AHR and TP53 are removed by the A allele, whereas binding sites for SOX18 and TP63 are created.

Renal phosphate and vitamin D metabolism is under the control of fibroblast growth factor 23 (FGF23), an endocrine and paracrine factor predominantly produced in bone. FGF23 formation is stimulated by active vitamin D, or parathyroid hormone (PTH), which are further regulators of phosphate homeostasis. In renal, inflammatory, and other diseases, plasma FGF23 reflects disease stage and correlates with outcome. Oncostatin M is part of the interleukin-6 (IL-6) family and regulates remodeling and PTH effects in bone as well as cardiac FGF23 production in heart failure via glycoprotein gp130. Here, we studied whether oncostatin M is a regulator of FGF23 in bone cells. Experiments were performed in UMR106 osteoblast-like cells, Fgf23 mRNA was determined by qRT-PCR, FGF23 protein by Western Blotting and ELISA, and oncostatin M receptor and leukemia inhibitory factor (LIF) receptor gene knockout accomplished by siRNA. As a result, oncostatin M dose-dependently up-regulated Fgf23 expression and protein secretion. The oncostatin M effect on FGF23 was mediated by oncostatin M receptor and gp130 and involved, at least in part, STAT3 and MEK1/2. Taken together, oncostatin M is a regulator of FGF23 through oncostatin M receptor, gp130, as well as STAT3 and MEK1/2 in UMR106 osteoblasts.

The NLRP3 inflammasome senses a variety of signals referred to as danger associated molecular patterns (DAMPs), including those triggered by crystalline particulates or degradation products of extracellular matrix. Since some DAMPs confer tissue-specific activation of the inflammasomes, we tested the hypothesis that bone matrix components function as DAMPs for the NLRP3 inflammasome and regulate osteoclast differentiation. Indeed, bone particles cause exuberant osteoclastogenesis in the presence of RANKL, a response that correlates with NLRP3 abundance and the state of inflammasome activation. To determine the relevance of these findings to bone homeostasis, we studied the impact of Nlrp3 deficiency on bone using pre-clinical mouse models of high bone turnover, including estrogen deficiency and sustained exposure to parathyroid hormone or RANKL. Despite comparable baseline indices of bone mass, bone loss caused by hormonal or RANKL perturbations is significantly reduced in Nlrp3 deficient than in wild type mice. Consistent with the notion that osteolysis releases DAMPs from bone matrix, pharmacologic inhibition of bone resorption by zoledronate attenuates inflammasome activation in mice. Thus, signals originating from bone matrix activate the NLRP3 inflammasome in the osteoclast lineage, and may represent a bone-restricted positive feedback mechanism that amplifies bone resorption in pathologic conditions of accelerated bone turnover.

NaPi-IIb/Slc34a2 is a Na+-dependent phosphate transporter that accounts for the majority of active phosphate transport into intestinal epithelial cells. Its abundance is regulated by dietary phosphate, being high during dietary phosphate restriction. Intestinal ablation of NaPi-IIb in mice leads to increased fecal excretion of phosphate, which is compensated by enhanced renal reabsorption. Here we compared the adaptation to dietary phosphate of wild type (WT) and NaPi-IIb-/- mice. High phosphate diet (HPD) increased fecal and urinary excretion of phosphate in both groups, though NaPi-IIb-/- mice still showed lower urinary excretion than WT. In both genotypes low dietary phosphate (LDP) resulted in reduced fecal excretion and almost undetectable urinary excretion of phosphate. Consistently, the expression of renal cotransporters after prolonged LDP was similar in both groups. Plasma phosphate declined more rapidly in NaPi-IIb-/- mice upon LDP, though both genotypes had comparable levels of 1,25(OH)2vitamin D3, parathyroid hormone and fibroblast growth factor 23. Instead, NaPi-IIb-/- mice fed LDP had exacerbated hypercalciuria, higher urinary excretion of corticosterone and deoxypyridinoline, lower bone mineral density and higher number of osteoclasts. These data suggest that during dietary phosphate restriction NaPi-IIb-mediated intestinal absorption prevents excessive demineralization of bone as an alternative source of phosphate.

Renal type II sodium-dependent inorganic phosphate (Pi) transporters NaPi2a and NaPi2c cooperate with other organs to strictly regulate the plasma Pi concentration. A high Pi load induces expression and secretion of the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) that enhance urinary Pi excretion and prevent the onset of hyperphosphatemia. How FGF23 secretion from bone is increased by a high Pi load and the setpoint of the plasma Pi concentration, however, are unclear. Here, we investigated the role of Transmembrane protein 174 (Tmem174) and observed evidence for gene co-expression networks in NaPi2a and NaPi2c function. Tmem174 is localized in the renal proximal tubules and interacts with NaPi2a, but not NaPi2c. In Tmem174-knockout (KO) mice, the serum FGF23 concentration was markedly increased but increased Pi excretion and hypophosphatemia were not observed. In addition, Tmem174-KO mice exhibit reduced NaPi2a responsiveness to FGF23 and PTH administration. Furthermore, a dietary Pi load causes marked hyperphosphatemia and abnormal NaPi2a regulation in Tmem174-KO mice. Thus, Tmem174 is thought to be associated with FGF23 induction in bones and the regulation of NaPi2a to prevent an increase in the plasma Pi concentration due to a high Pi load and kidney injury.

The objective of this study was to determine the impact of calcium sensing receptor (CASR) A990G genetic polymorphism on parathyroid hormone (PTH) lowering response to cinacalcet treatment when controlling for significant influencing clinical factors. This retrospective study was conducted on 135 Thai hemodialysis (HD) patients with secondary hyperparathyroidism (SHPT). CASR A990G genotypes were determined. The patients were identified as either G carriers (heterozygous or homozygous CASR 990G allele carriers) or noncarriers (homozygous CASR 990A carriers). Tested covariates were baseline PTH level (bPTH), baseline serum phosphate (bPhos), baseline serum calcium (bCa), baseline calcitriol equivalent dose (bCtriol), baseline ergocalciferol dose (bErgo), and age. The ANCOVA showed that intact PTH levels after 12 weeks of cinacalcet treatment (PTHw12) was significantly lower among G carriers compared with noncarriers after controlling for bPTH, bPhos, bCtriol, and bErgo (F(1, 127) = 15.472, p < 0.001), with the adjusted mean difference of 253.7 pg/mL. The logistic regression analysis revealed that the odds of a G carrier achieving 30% PTH reduction after 12-week cinacalcet treatment were 3.968 times greater than the odds for a noncarrier after adjusting for bPhos, bCtriol, and age. In conclusion, the CASR A990G polymorphism significantly influences cinacalcet response in HD patients with SHPT.

We have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (- 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.

Intermittent Parathyroid Hormone (I-PTH) is the only FDA approved anabolic drug therapy available for the treatment of osteoporosis in males and postmenopausal females. The effects of I-PTH on the chondrogenic lineage of the mandibular condylar cartilage (MCC) are not well understood. To investigate the role of I-PTH on the MCC and subchondral bone, we carried out our studies using 4 to 5 week old triple transgenic mice (Col1a1XCol2a1XCol10a1). The experimental group was injected with PTH (80 μg/kg) daily for 2 weeks, while control group was injected with saline. Our histology showed that the I-PTH treatment led to an increased number of cells expressing Col1a1, Col2a1 and Col10a1. Additionally, there was an increase in cellular proliferation, increased proteoglycan distribution, increased cartilage thickness, increased TRAP activity, and mineralization. Immunohistochemical staining showed increased expression of pSMAD158 and VEGF in the MCC and subchondral bone. Furthermore our microCT data showed that I-PTH treatment led to an increased bone volume fraction, tissue density and trabecular thickness, with a decrease in trabecular spacing. Morphometric measurements showed increased mandibular length and condyle head length following I-PTH treatment. In conclusion, our study suggests that I-PTH plays a critical role in cellular proliferation, proteoglycan distribution, and mineralization of the MCC.

The purpose of this study was to investigate the effect of administering intermittent parathyroid hormone (iPTH) before tooth extraction versus after tooth extraction on the risk of developing MRONJ in experimental animal model. Twenty-five ovariectomized rats received 6 weeks of bisphosphonate therapy. They were classified into 3 groups, based on the timing of the medication, as Control, Pre-PTH and Post-PTH groups. For Control group, normal saline was administered before and after tooth extraction. iPTH was administered during 4 weeks before tooth extraction for Pre-PTH group and after tooth extraction for Post-PTH group. The animals were euthanized 8 weeks after tooth extraction. Macroscopic, histological, micro-computed tomography (micro-CT), and histomorphometric examinations were conducted. The incidences of impaired healing were 11.11% both in Pre-PTH and Post-PTH groups, which was lower than the Control group (42.86%). Bone healing in the extraction socket, based on micro-CT and histomorphometry evaluations, was best in Post-PTH and worst in Control group. The Pre-PTH group showed moderate healing pattern. Despite of limitations in this study, the authors identified Pre-PTH group seems to have positive effect on extraction socket healing. With regard to timing, administering iPTH after tooth extraction was superior to applying it before tooth extraction.

Bone marrow ablation prompts transient bone formation in nearly the entire medullary cavity before marrow regeneration occurs. Here, we establish a procedure to direct bone formation in a desired particular site within the medullary cavity for support of biomedical devices. Local intramedullary injury was performed in the tibiae of rats and parathyroid hormone (PTH), alendronate, or saline was administered. Newly generated bone in the medulla was assessed by micro-CT and histology. To evaluate the function of newly generated bone, animals received intramedullary injury in tibiae followed by daily PTH. At day-14, implants were placed in the endocortical bone and the bone response to the implants was assessed. The fate of newly generated bone was compared with and without implants. We found that neither intramedullary injury nor medication alone resulted in bone formation. However, when combined, substantial bone was generated locally inside the diaphyseal medulla. Newly formed bone disappeared without implant placement but was retained with implants. Bone was especially retained around and between the implants. This study found that local bone marrow disruption followed by PTH or alendronate generated substantial cancellous bone locally in the diaphyseal medulla. This approach offers promise as a tissue engineering tool in medicine and dentistry.

Vascular calcification is a component of cardiovascular disease, which is leading cause of death in patients with chronic kidney disease (CKD). A functional assay (T50-test) measuring the propensity of human serum to calcify associates with mortality and cardiovascular events in CKD patients. Calcification propensity is known to increase with CKD stage. We investigated whether the T50 readout is directly dependent on excretory kidney function (eGFR) or rather explained by deranged parameters of bone and mineral metabolism in the course of CKD. T50, along with markers implicated in calcification and mineral metabolism, were measured in a cross-sectional cohort of 118 patients with CKD stage 1-5. Associations of T50 with measured parameters were analysed and partial correlations performed to test to which extent the association of T50 with eGFR can be attributed to variation of these parameters. T50 correlates with eGFR, but serum levels of phosphate and calcium largely explain this association. Phosphate, magnesium, fetuin A, albumin, bicarbonate, and serum cross-laps but not Parathyroid Hormone or Fibroblast Growth Factor 23 are associated with T50 in multivariate adjusted models. These findings indicate that T50 values depend mainly on the concentration of promoters and inhibitors of calcification in serum, but not excretory kidney function.

The aims of this randomized controlled double-blind clinical trial were to assess the impact of vitamin D supplementation on calcium metabolism and non-calcemic broad gene expression by relating them to the individual's responsiveness to varying doses of vitamin D3. Thirty healthy adults were randomized to receive 600, 4,000 or 10,000 IU/d of vitamin D3 for 6 months. Circulating parathyroid hormone (PTH), 25(OH)D, calcium and peripheral white blood cells broad gene expression were evaluated. We observed a dose-dependent increase in 25(OH)D concentrations, decreased PTH and no change in serum calcium. A plateau in PTH levels was achieved at 16 weeks in the 4000 and 10,000 IU/d groups. There was a dose-dependent 25(OH)D alteration in broad gene expression with 162, 320 and 1289 genes up- or down-regulated in their white blood cells, respectively. Our results clearly indicated that there is an individual's responsiveness on broad gene expression to varying doses of vitamin D3. Vitamin D3 supplementation at 10,000 IU/d produced genomic alterations several fold higher than 4,000 IU/d even without further changes in PTH levels. Our findings may help explain why there are some inconsistency in the results of different vitamin D's clinical trials.

Kidneys are key regulators of phosphate homeostasis. Biallelic mutations of the renal Na+/phosphate cotransporter SLC34A1/NaPi-IIa cause idiopathic infantile hypercalcemia, whereas monoallelic mutations were frequently noted in adults with kidney stones. Genome-wide-association studies identified SLC34A1 as a risk locus for chronic kidney disease. Pathogenic mutations in SLC34A1 are present in 4% of the general population. Here, we characterize a mouse model carrying the 91del7 in-frame deletion, a frequent mutation whose significance remains unclear. Under normal dietary conditions, 12 weeks old heterozygous and homozygous males have similar plasma and urinary levels of phosphate as their wild type (WT) littermates, and comparable concentrations of parathyroid hormone, fibroblast growth factor 23 (FGF-23) and 1,25(OH)2 vitamin D3. Renal phosphate transport, and expression of NaPi-IIa and NaPi-IIc cotransporters, was indistinguishable in the three genotypes. Challenging mice with low dietary phosphate did not result in differences between genotypes with regard to urinary and plasma phosphate. Urinary and plasma phosphate, plasma FGF-23 and expression of cotransporters were similar in all genotypes after weaning. Urinary phosphate and bone mineral density were also comparable in 300 days old WT and mutant mice. In conclusion, mice carrying the 91del7 truncation do not show signs of impaired phosphate homeostasis.

Cardiac valve calcification is highly prevalent in patients with chronic kidney disease (CKD). Low vitamin D levels are associated with vascular calcification in CKD. However, the association between vitamin D levels and cardiac valve calcification is unknown. A total of 513 patients with pre-dialysis CKD were included in this cross-sectional study. Aortic valve calcification (AVC) and mitral valve calcification (MVC) were assessed using two-dimensional echocardiography. The associations between AVC and MVC and baseline variables were investigated using logistic regression analyses. In multivariable analysis, serum 1,25(OH)2D level was independently associated with AVC (odds ratio [OR], 0.87; P < 0.001) and MVC (OR, 0.92; P < 0.001). Additionally, age, diabetes, coronary heart disease, calcium × phosphate product, and intact parathyroid hormone levels were independently associated with AVC and MVC. Systolic blood pressure was independently associated with AVC. A receiver-operating characteristic (ROC) curve analysis showed that the best cutoff values of serum 1,25(OH)2D levels for predicting AVC and MVC were ≤ 12.5 and ≤ 11.9 pg/dl, respectively. Serum 1,25(OH)2D deficiency is independently associated with AVC and MVC in patients with CKD, suggesting that serum 1,25(OH)2D level may be a potential biomarker of AVC and MVC in these patients.

Fibroblast growth factor 23, parathyroid hormone, and 1,25-dihydroxyvitamin D are critical in phosphate homeostasis. Despite these factors' importance, regulators of phosphaturia in the acute postprandial phase remain largely unknown. This study investigated the mechanism of acute phosphate regulation in the postprandial phase in rats. Duodenal administration of radiolabeled phosphate (32P) showed that 32P levels in the inferior vena cava (IVC) blood were lower than those in the portal vein (PV) blood. Serum phosphate concentration transiently increased 5 min after phosphate solution administration through IVC, while it was maintained after the administration through PV. Phosphate administration through both IVC and PV resulted in increased fractional excretion of phosphate (FEPi) at 10 min without elevation of the known circulating factors, but urinary phosphate excretion during the period was 8% of the dose. Experiments using 32P or partial hepatectomy showed that the liver was one of the phosphate reservoirs. The elevation of FEPi and suppression of sodium-phosphate cotransporter 2a in the kidney at 10 min was attenuated in rats with SCH23390, hepatic denervation, or renal denervation, thus indicating that the liver communicated with the kidney via the nervous system to promote phosphaturia. These results revealed previously unknown mechanisms for serum phosphate maintenance.

Allolobophora calignosa (Ac) is a folk medicine for millennia, as it possesses many biological activities. This study aimed to investigate the chemo-preventive activity of A.calignosa coelomic fluid (AcCF) and A.calignosa extract (AcE) on glucocorticoid-induced osteoporosis (GIOP) in mice. Characterization and in vitro biological activity of AcE and AcCF has been assessed. Male CD-1 mice were subcutaneously received dexamethasone (DEX) (1 mg/kg, 5 times/week) and concurrently intraperitoneally treated with either AcCF (20 mg/kg) or AcE (45 mg/kg) every other day for 28 days. Serum and bone homogenates were subjected for qPCR and biochemical analysis. AcE and AcCF treatment significantly increased bone mineral density (BMD), bone mineral content (BMC), calcium (Ca), phosphorus (P), and calcitonin levels, whereas activity of serum alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), serum acidic phosphatase (ACP), bone acidic phosphatase (BACP) and parathyroid hormone (PTH) levels were significantly reduced compare with untreated GIOP mice. Treatment with AcE and AcCF modulates oxidative stress and downregulated Rank and Mmp9 expression, as well as increased glycosaminoglycan content in the organic bone matrix, resulting in osteoclastogenesis inhibition. Overall, AcCF and AcE show a chemo-preventive activity against GIOP by inhibiting oxidative stress and regulating expression and/or activity of osteoblast/osteoclast-related markers.

Our research found that vitamin D3 (VD3) treatment increased lung metastasis in mice with 4T1 murine breast cancer (BC). This study aims to investigate the impact of VD3 on the activation of tumor-associated macrophages (TAMs) in BC. Mice bearing 4T1, E0771, 67NR BC cells, and healthy mice, were fed diets with varying VD3 contents (100-deficient, 1000-normal, and 5000 IU/kg-elevated). Some mice in the 1000 and 100 IU/kg groups received calcitriol. We studied bone metastasis and characterized TAMs and bone marrow-derived macrophages (BMDMs). 4T1 cells had higher bone metastasis potential in the 5000 IU/kg and calcitriol groups. In the same mice, an elevated tumor osteopontin level and M2 polarization of TAMs (MHCIIlow CD44high phenotype) were observed. Gene expression analysis confirmed M2 polarization of 4T1 (but not 67NR) TAMs and BMDMs, particularly in the 100 IU + cal group (increased Mrc1, Il23, and Il6). This polarization was likely due to COX-2/PGE2 induction in 4T1 calcitriol-treated cells, leading to increased proinflammatory cytokines like IL-6 and IL-23. Future studies will explore COX-2/PGE2 as a primary mediator of calcitriol-stimulated inflammation in the BC microenvironment, especially relevant for BC patients with VD3 deficiency and supplementation.