Dietary Potassium Supplementation Reduces Chronic Kidney Lesions Independent of Blood Pressure in Deoxycorticosterone-Acetate and High Sodium Chloride-Treated Mice.

We have previously shown that an excess of deoxycorticosterone acetate and high sodium chloride intake (DOCA/salt) in one-renin gene mice induces a high urinary Na/K ratio, hypokalemia, and cardiac and renal hypertrophy in the absence of hypertension. Dietary potassium supplementation prevents DOCA/salt-induced pathological processes. In the present study, we further study whether DOCA/salt-treated mice progressively develop chronic inflammation and fibrosis in the kidney and whether dietary potassium supplementation can reduce the DOCA/salt-induced renal pathological process. Results showed that (1) long-term DOCA/salt-treated one-renin gene mice developed severe kidney injuries including tubular/vascular hypertrophy, mesangial/interstitial/perivascular fibrosis, inflammation (lymphocyte's immigration), proteinuria, and high serum creatinine in the absence of hypertension; (2) there were over-expressed mRNAs of plasminogen activator inhibitor-1 (PAI-1), fibronectin, collagen type I and III, interferon-inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP1), transforming growth factor-ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), osteopontin, Nuclear factor kappa B (NF-κB)/P65, and intercellular adhesion molecule (ICAM)-1; and (3) dietary potassium supplementation normalized urinary Na/K ratio, hypokalemia, proteinuria, and serum creatinine, reduced renal hypertrophy, inflammations, and fibrosis, and down-regulated mRNA expression of fibronectin, Col-I and III, TGF-ß, TNF-α, osteopontin, and ICAM without changes in the blood pressure. The results provide new evidence that potassium and sodium may modulate proinflammatory and fibrotic genes, leading to chronic renal lesions independent of blood pressure.

Secreted phosphoprotein 1 regulates natural compound 3',4',5,7-tetrahydroxyflavone to inhibit mast cell-mediated allergic inflammation.

BACKGROUND: Mast cells (MCs) are important effector cells in anaphylaxis and anaphylactic disease. 3',4',5,7-tetrahydroxyflavone (THF) presents in many medicinal plants and exerts a variety of pharmacological effects. In this study, we evaluated the effect of THF on C48/80-induced anaphylaxis and the mechanisms underlying its effects, including the role of secreted phosphoprotein 1 (SPP1), which has not been reported to IgE-independent MC activation. RESULTS: THF inhibited C48/80-induced Ca2+ flow and degranulation via the PLCγ/PKC/IP3 pathway in vitro. RNA-seq showed that THF inhibited the expression of SPP1 and downstream molecules. SPP1 is involved in pseudo-anaphylaxis reactions. Silencing SPP1 affects the phosphorylation of AKT and P38. THF suppressed C48/80-induced paw edema, hypothermia and serum histamine, and chemokines release in vivo. CONCLUSIONS: Our results validated SPP1 is involved in IgE-independent MC activation anaphylactoid reactions. THF inhibited C48/80-mediated anaphylactoid reactions both in vivo and in vitro, suppressed calcium mobilization and inhibited SPP1-related pathways.

Low concentrations of grape seed extract maintain osteoblast morphology, cell adhesion, and mineralization.

The increase in life expectancy has led to a higher incidence of osteoporosis, characterized by an imbalance in bone remodeling. Several drugs are used for its treatment, but most promote undesirable side effects. The present investigation evaluated the effects of two low concentrations of grape seed extract (GSE) rich in proanthocyanidins on MC3T3-E1 osteoblastic cells. The cells were cultured in an osteogenic medium and divided into control (C), 0.1 µg/mL GSE (GSE0.1), and 1.0 µg/mL GSE (GSE1.0) groups to evaluate cell morphology, adhesion, and proliferation, in situ alkaline phosphatase (ALP) detection, mineralization and immunolocalization of osteopontin (OPN). The data obtained were analyzed by statistical tests for a significance of 5%. Cell morphology was maintained with both GSE concentrations, whereas cell adhesion significantly increased within three days in all groups. Cell proliferation increased significantly at seven days of culture, followed by a significant decrease in all experimental periods, with no statistical difference among them. In situ detection of ALP and mineralization increased with time, but within each period, no statistical differences among groups were observed. The expression of osteopontin was distributed regularly with more intensity after 24 hours in the GSE0.1 group. After three days, OPN expression was more intense in the control group, followed by GSE0.1 and GSE1.0 groups. Data obtained suggest that low concentrations of GSE do not affect the morphology and may stimulate the functional activity of osteoblastic cells.

Exosomal STAT1 derived from high phosphorus­stimulated vascular endothelial cells induces vascular smooth muscle cell calcification via the Wnt/ß­catenin signaling pathway.

Vascular calcification is commonly observed in chronic kidney disease. The mechanism of how the calcification signal from endothelial cells is transmitted to vascular smooth muscle cells (VSMCs) remains unknown. The aim of the present study was to investigate whether exosomes from HUVECs (HUVEC­Exos) could regulate VSMC calcification and its potential signaling pathway. HUVEC­Exos were isolated from HUVECs under no phosphorus (NP) and high phosphorus (HP) conditions. Alizarin Red S staining and calcium (Ca) content analysis were carried out to detect calcification in VSMCs. Proteomics analysis was carried out to detect the differential expression of exosomal proteins. Protein and mRNA levels were measured by western blot analysis and reverse transcription­quantitative PCR (RT­qPCR). Exosomes derived from HP­HUVECs promoted the calcification of VSMCs, as assessed by Alizarin Red S staining, alkaline phosphatase activity assays, Ca content measurements and the increased expression of runt­related transcription factor 2 and osteopontin. Proteomic analysis detected the upregulation of STAT1 in HP­exosomes from HUVECs (HUVEC­Exos) compared with NP­HUVEC­Exos, which was also confirmed by western blot analysis and RT­qPCR. Inhibition of STAT1 expression in VSMCs using fludarabine or knockdown of STAT1 expression using small interfering RNA alleviated the calcification of VSMCs. Furthermore, lithium chloride (Wnt activator) reversed the protective effect of STAT1 inhibition on VSMC calcification, while Dickkopf­1 (Wnt inhibitor) exerted the opposite effect, suggesting that activation of the Wnt/ß­catenin signaling pathway was involved in STAT1­mediated VSMC calcification. In conclusion, the present results indicated that exosomal STAT1 derived from HP­treated HUVECs could promote VSMC calcification, and activation of the Wnt/ß­catenin pathway may be a potential mechanism of the VSMC calcification promoted by exosomes.

A Tissue Engineering Acoustophoretic (TEA) Set-up for the Enhanced Osteogenic Differentiation of Murine Mesenchymal Stromal Cells (mMSCs).

Quickly developing precision medicine and patient-oriented treatment strategies urgently require novel technological solutions. The randomly cell-populated scaffolds usually used for tissue engineering often fail to mimic the highly anisotropic characteristics of native tissue. In this work, an ultrasound standing-wave-based tissue engineering acoustophoretic (TEA) set-up was developed to organize murine mesenchymal stromal cells (mMSCs) in an in situ polymerizing 3-D fibrin hydrogel. The resultant constructs, consisting of 17 cell layers spaced at 300 µm, were obtained by continuous wave ultrasound applied at a 2.5 MHz frequency. The patterned mMSCs preserved the structured behavior within 10 days of culturing in osteogenic conditions. Cell viability was moderately increased 1 day after the patterning; it subdued and evened out, with the cells randomly encapsulated in hydrogels, within 21 days of culturing. Cells in the structured hydrogels exhibited enhanced expression of certain osteogenic markers, i.e., Runt-related transcription factor 2 (RUNX2), osterix (Osx) transcription factor, collagen-1 alpha1 (COL1A1), osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), as well as of certain cell-cycle-progression-associated genes, i.e., Cyclin D1, cysteine-rich angiogenic inducer 61 (CYR61), and anillin (ANLN), when cultured with osteogenic supplements and, for ANLN, also in the expansion media. Additionally, OPN expression was also augmented on day 5 in the patterned gels cultured without the osteoinductive media, suggesting the pro-osteogenic influence of the patterned cell organization. The TEA set-up proposes a novel method for non-invasively organizing cells in a 3-D environment, potentially enhancing the regenerative properties of the designed anisotropic constructs for bone healing.

A molecular phenotypic screen reveals that lobetyolin alleviates cardiac dysfunction in 5/6 nephrectomized mice by inhibiting osteopontin.

BACKGROUND: Cardiovascular diseases are the major cause of mortality in patients with advanced chronic kidney diseases. The predominant abnormality observed among this population is cardiac dysfunction secondary to myocardial remodelings, such as hypertrophy and fibrosis, emphasizing the need to develop potent therapies that maintain cardiac function in patients with end-stage renal disease. AIMS: To identify potential compounds and their targets as treatments for cardiorenal syndrome type 4 (CRS) using molecular phenotyping and in vivo/in vitro experiments. METHODS: Gene expression was assessed using bioinformatics and verified in animal experiments using 5/6 nephrectomized mice (NPM). Based on this information, a molecular phenotyping strategy was pursued to screen potential compounds. Picrosirius red staining, wheat germ agglutinin staining, Echocardiography, immunofluorescence staining, and real-time quantitative PCR (qPCR) were utilized to evaluate the effects of compounds on CRS in vivo. Furthermore, qPCR, immunofluorescence staining and flow cytometry were applied to assess the effects of these compounds on macrophages/cardiac fibroblasts/cardiomyocytes. RNA-Seq analysis was performed to locate the targets of the selected compounds. Western blotting was performed to validate the targets and mechanisms. The reversibility of these effects was tested by overexpressing Osteopontin (OPN). RESULTS: OPN expression increased more remarkably in individuals with uremia-induced cardiac dysfunction than in other cardiomyopathies. Lobetyolin (LBT) was identified in the compound screen, and it improved cardiac dysfunction and suppressed remodeling in NPM mice. Additionally, OPN modulated the effect of LBT on cardiac dysfunction in vivo and in vitro. Further experiments revealed that LBT suppressed OPN expression via the phosphorylation of c-Jun N-terminal protein kinase (JNK) signaling pathway. CONCLUSIONS: LBT improved CRS by inhibiting OPN expression through the JNK pathway. This study is the first to describe a cardioprotective effect of LBT and provides new insights into CRS drug discovery.

Gallic acid ameliorates calcium oxalate crystal-induced renal injury via upregulation of Nrf2/HO-1 in the mouse model of stone formation.

BACKGROUND: High prevalence and reoccurrence rate of nephrolithiasis bring about serious socioeconomic and healthcare burden, necessitating the need of effective therapeutic agents. Previous study revealed that gallic acid (GAL) alters the nucleation pathway of calcium oxalate (CaOx). On the other hand, it appears protective role against oxidative injury. Whether GAL could protect against crystal-induced lesion in vivo, and its underlying mechanism is yet unsolved. PURPOSE: This study aims to investigate the protective effects of GAL on the crystal-induced renal injury and its underlying mechanism in the mouse model of stone formation induced by glyoxylic acid. STUDY DESIGN AND METHODS: The mouse model of stone formation was established via successive intraperitoneal injection of glyoxylate. Proximal tubular epithelial cell line HK-2 treated with calcium oxalate monohydrate (COM) was used as in vitro model. The protective role of GAL on nephrolithiasis was tested by determination of tubular injury, crystal deposition and adhesion, levels of inflammatory cytokines, macrophage infiltration and the redox status of kidney. In vitro, effect of GAL on the ROS level and oxidative tubular injury induced by COM were detected, as well as major antioxidant pathway Nrf2/HO-1. RESULTS: Administration of GAL alleviates the renal deposition and adhesion of CaOx stone. Meanwhile, GAL ameliorates the inflammation and renal tubular injury. Level of intracellular ROS, osteopontin and CD44 are reduced, either in the mouse model of stone formation or in the COM-treated HK-2 cells after treatment of GAL. Mechanistically, GAL activates Nrf2/HO-1 pathway in HK-2 cells. Silencing Nrf2 abrogates the protective effect of GAL on the oxidative injury and adhesion of COM in HK-2 cells. CONCLUSION: Taken together, our study demonstrates the protective effect of GAL on the deposition of kidney stone and consequent tubular injury. Induction of the antioxidant pathway Nrf2/HO-1 was found to decrease the level of ROS and oxidative injury, thus implying that GAL could be a potential therapeutic agent for the treatment of nephrolithiasis.

Downregulation of inflammatory mediators by ethanolic extract of Bergenia ligulata (Wall.) in oxalate injured renal epithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In the Indian traditional system of medicine, Bergenia ligulata (Wall.) Engl. has been used for treatment of urolithiasis. Its efficacious nature has led to its incorporation in various commercial herbal formulations such as Cystone and Neeri which are prescribed for kidney related ailments. AIM OF THE STUDY: To assess whether ethanolic extract of B. ligulata can mitigate the cascade of inflammatory responses that cause oxidative stress and ultimately cell death in renal epithelial cells exposed to hyperoxaluric conditions. MATERIAL AND METHODS: Bioactivity guided fractionation using solvents of varying polarities was employed to evaluate the potential of the extracts of B. ligulata to inhibit the crystallization process. Modulation of crystal morphology was visualized through Scanning electron microscopy (SEM) analysis. Cell death was assessed using flow cytometry based assays. Alteration in the inflammatory mediators was evaluated using real time PCR and immunocytochemistry. Phytochemical characterization of the ethanolic extract was carried out using FTIR, LC-MS and GC-MS. RESULTS: Bioactivity guided fractionation for the assessment of antilithiatic activity revealed dose dependent inhibition of nucleation and aggregation process of calcium oxalate crystals in the presence of various extracts, however ethanolic extract showed maximum inhibition and was chosen for further experiments. Studies on renal epithelial NRK-52E cells showed, cytoprotective efficacy of B. ligulata extract against oxalate injury. SEM anaysis further revealed the potential of the extract to modulate the crystal structure and adhesion to renal cell surface. Exposure of the renal cells to the extract led to conversion of the calcium oxalate monohydrate (COM) crystals to the less injurious calcium oxalate dihydrate (COD) form. Expression analysis for oxidative stress and inflammatory biomarkers in NRK-52E cells revealed up-regulation of Mitogen activated protein kinase (MAPK), Osteopontin (OPN) and Nuclear factor- ĸB (NF-ĸB), in response to calcium oxalate insult; which was drastically reduced in the presence of B. ligulata extract. Flow cytometric evaluation pointed to caspase 3 mediated apoptotic cell death in oxalate injured cells, which was attenuated by B. ligulata extract. CONCLUSION: Considering the complex multifactorial etiology of urolithiasis, ethanolic extract from B. ligulata can be a promising option for the management of kidney stones, as it has the potential to limit inflammation and the subsequent cell death.

MicroRNA-409-3p promotes osteoblastic differentiation via activation of Wnt/ß-catenin signaling pathway by targeting SCAI.

Osteogenic differentiation is an important process of new bone formation, microRNA-409-3p (miR-409-3p) has been reported to be up-regulated in the osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The present study aimed to investigate the regulatory effect of miR-409-3p on osteogenic differentiation of MSCs and its molecular mechanism. The expression of miR-409-3p in osteoblast (human skull osteoblast, HCO) and bone marrow-derived MSCs (MSC-A, MSC-B, MSC-U) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding of miR-409-3p to suppressor of cancer cell invasion (SCAI) in MSC-B was investigated by performing a dual-luciferase reporter gene assay. MSC-B was selected to transfect with miR-409-3p analog/complementary sequence (cs), miR-409-3p analog + SCAI and miR-409-3p cs + small interfering (si)-SCAI, as well as control, respectively. The alkaline phosphatase (ALP) activity, Alizarin Red staining, and the expression of osteogenic markers (ALP, osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RUNX2)) in MSC-B during osteoblastic differentiation were tested by RT-qPCR and Western blotting, respectively. Additionally, the Wnt/ß-catenin pathway was inhibited by dickkopf-related protein 1 (DKK-1) to get the roles of miR-409-3p during the osteoblastic differentiation of MSC-B when transfected with miR-409-3p analog. The expression of miR-409-3p in HCO was higher than that in these three MSCs and showed an increasing time-dependent trend on the 0 and 21st day of osteoblastic differentiation. MiR-409-3p directly regulated SCAI by targeting SCAI 3'UTR. Further, miR-409-3p suppressed SCAI expression, but SCAI up-regulation suppressed the osteoblastic differentiation, as well as reduced the relative mRNA/protein expression of Wnt/ß-catenin signaling pathway-related genes (Axis inhibition protein 1 (AXIN1), ß-catenin, Lymphoid Enhancer Binding Factor 1, Cellular-myelocytomatosis (c-myc) and cyclin D1). Importantly, disruption of Wnt signaling also blocked miR-409-3p induced osteoblastic differentiation of MSCs. Therefore, miR-409-3p promotes osteoblastic differentiation through the activation of the Wnt/ß-catenin pathway by down-regulating SCAI expression.

Ginkgobilol, a new diarylpentanoid and an osteogenic diarylpentanoid analog from Ginkgo biloba leaves.

Phytochemical analysis of methanol extracts of Ginkgo biloba leaves resulted in the isolation of a novel diarylpentanoid, ginkgobilol (1) and a known diarylpentanoid analog (2). The structure of the new compound was elucidated by analyzing NMR spectroscopic data and HR-ESIMS, and the absolute configuration was determined using gauge-including atomic orbital NMR chemical shift calculations, followed by DP4+ analysis and specific rotation value. Diarylpentanoids comprise two aromatic rings linked by a five-carbon bridge; these are relatively unique examples in natural products. To the best of our knowledge, the present study is the first to report the presence of diarylpentanoids in G. biloba. Compound 2 increased alkaline phosphatase (ALP) production in C3H10T1/2, a murine mesenchymal stem cell line, in a dose-dependent manner. The promotion of osteogenic differentiation by the active compound 2 mediated by induction of transcriptional ALP and osteopontin (OPN) gene expression was confirmed using quantitative real time polymerase chain reaction, thus indicating its remarkable bone formation activity.

A high-fat diet rich in corn oil exaggerates the infarct size and memory impairment in rats with cerebral ischemia and is associated with suppressing osteopontin and Akt, and activating GS3Kß, iNOS, and NF-κB.

The increase in osteopontin (OPN) levels after stroke induces neural protection by activating Akt signaling and inhibiting GS3Kß, iNOS, and NF-κB. This study investigated the effect of a high-fat diet rich in corn oil (CO-HFD) on infarct size and memory function in rats after induction of cerebral ischemia in rats and investigated its effect on the expression of OPN/Akt/iNOS/NF-κB signaling pathways. Rats were initially fed a standard diet (STD, 3.82 kcal/g; 9.4%, from fat) or a CO-HFD (5.4 kcal/g, 40% from fat) for 12 weeks. Then, both groups were further subdivided into either sham group or group exposed to cerebral ischemia by the middle cerebral artery occlusion (MCAO) protocol. Compared with sham-operated rats fed STD diet, neurological scores and both short- and long-term memory functions were significantly impaired in sham-operated CO-HFD-fed rats. In addition, brains collected from CO-HFD-fed rats showed lower protein levels of OPN, p-Akt (Thr308), p-GS3Kß (Ser9), and Bcl-2 and had higher protein levels of iNOS, cleaved caspase-3, nuclear NF-κB p65, and cytoplasmic cytochrome C. However, once exposed to MCAO surgery, similar but more profound alterations of all these biochemical parameters with more severe impairment in short- and long-term memory functions and larger infarct size were noticed in the brains of CO-HFD-fed rats as compared with STD-fed rats exposed to MCAO. In conclusion, chronic consumption of CO-HFD induces memory impairments and worsens memory function recovery and infarct size after cerebral ischemia in rats by reducing levels of OPN, inhibiting the activation of Akt and activating iNOS and NF-κB.

Polymeric nanofibrous scaffolds laden with cell-derived extracellular matrix for bone regeneration.

Bone tissue engineering aims to alleviate the shortage of available autograft material and the biological/mechanical incompatibility of allografts through fabrication of bioactive synthetic bone graft substitutes. However, these substitute grafting materials have insufficient biological potency that limits their clinical efficacy in regenerating large defects. Extracellular matrix, a natural tissue scaffold laden with biochemical and structural cues regulating cell adhesion and tissue morphogenesis, may be a versatile supplement that can extend its biological functionality to synthetic grafts. Embedding decellularized extracellular matrix (dECM) into synthetic polymers offers a promising strategy to enhance cellular response to synthetic materials, mitigate physical and mechanical limitations of dECMs, and improve clinical utility of synthetic bone grafts. Enriched with dECM biochemical cues, synthetic polymers can be readily fabricated into complex biocomposite grafts that mimic bone structure and stimulate endogenous cells to regenerate bone. In this study, cell-derived dECMs from osteoblast and endothelial cells were incorporated into polycaprolactone (PCL) solutions for electrospinning dual-layer nanofibrous scaffolds with osteogenic and vascular cues. The study examined the bioactivity of dECM scaffolds in osteoblast cultures for cell number, mineral deposits, and osteogenic markers, as well as regeneration of cortical bone defect in a rat femur. Scaffolds with osteoblast dECM had a significantly robust osteoblast proliferation, Alizarin Red staining/concentration, and osteopontin-positive extracellular deposits. Implanted scaffolds increased bone growth in femoral defects, and constructs with both osteogenic and vascular cues significantly improved cortical width. These findings demonstrate the potential to fabricate tailored biomimetic grafts with dECM cues and fibrous architecture for bone applications.

Sulfurous thermal waters stimulate the osteogenic differentiation of human mesenchymal stromal cells - An in vitro study.

Strategies aimed at delaying the onset of bone tissue degeneration and the resulting skeletal fragility are key to decrease the risk of bone fracture correlated to ageing. The therapeutic properties of sulfurous thermal waters (STWs), rich in hydrogen sulfide (H2S), have been claimed for centuries. However, the direct regulation of bone cells by STWs has not been investigated yet. Here we aimed at analyzing the effect of STWs on cultured human mesenchymal stromal cells (hMSCs) derived from bone tissue. Two concentrations of STWs from 2 health spa centers in Italy (here named STW-1 and STW-2) containing, respectively, high and moderate quantities of H2S, were added to the culture media. Cytotoxicity and osteogenic differentiation were evaluated. We provided first evidence that treatment of hMSCs with STWs results in a sharp increase in intracellular H2S content, coherent with the different concentrations of H2S, thereby reveling that STWs-released H2S is internalized by cells. STWs treatment significantly induced osteogenic differentiation of hMSCs. In particular, mineral apposition was increased with a similar pattern by the two STWs, while mRNA expression of osteogenic markers (BSP, OC, RUNX-2, OPN) was differently affected. Only STW-2 induced a significant, dose-dependent increase in these gene expression. These findings support the rationale for the use of STWs as a complementary treatment of bone wasting diseases.

Preprotection of Tea Polysaccharides with Different Molecular Weights Can Reduce the Adhesion between Renal Epithelial Cells and Nano-Calcium Oxalate Crystals.

Crystal adhesion is an important link in the formation of kidney stones. This study investigated and compared the adhesion differences between nano-calcium oxalate monohydrate (COM) and human renal proximal tubule epithelial (HK-2) cells before and after treatment with tea polysaccharides (TPSs) TPS0, TPS1, TPS2, and TPS3 with molecular weights of 10.88, 8.16, 4.82, and 2.31 kDa, respectively. TPS treatment effectively reduced the damage of COM to HK-2 cells, thereby resulting in increased cell activity, decreased release of lactate dehydrogenase, cell morphology recovery, decreased level of reactive oxygen species, increased mitochondrial membrane potential, increased lysosomal integrity, decreased expression of adhesion molecule osteopontin and eversion of phosphatidylserine, and decreased crystal adhesion. Among the TPSs, TPS2 with moderate molecular weight had the best protective effect on cells and the strongest effect on the inhibition of crystal adhesion. Thus, TPS2 may be a potential anticalculus drug.

Seaweed Porphyra yezoensis polysaccharides with different molecular weights inhibit hydroxyapatite damage and osteoblast differentiation of A7R5 cells.

Vascular calcification (VC) is a common pathological manifestation in patients with cardiovascular diseases, leading to high mortality in patients with chronic kidney diseases. The deposition of hydroxyapatite (HAP) crystals on vascular smooth muscle cells leads to cell damage, which promotes osteogenic transformation. In this study, four different molecular weights (MWs ) of Porphyra yezoensis polysaccharides (PYP1, PYP2, PYP3, and PYP4 with MWs of 576, 49.5, 12.6, and 4.02 kDa, respectively) were used to coat HAP, and the differences in toxicity and calcification of HAP on A7R5 cells before and after coating were studied. The results showed that PYPs could effectively reduce HAP damage to the A7R5 cells. Under the protection of PYPs, cell viability increased and lactate dehydrogenase release, active oxygen level, and cell necrosis rate decreased; also, the amount of the HAP crystals adhering to cell surfaces and entering cells decreased. PYPs with low molecular weights presented better protective effects than high-molecular-weight PYPs. PYPs also inhibited the osteogenic transformation of the A7R5 cells induced by HAP and decreased alkaline phosphatase (ALP) activity and expressions of bone/chondrocyte phenotype genes (runt-related factor 2, ALP, osteopontin, and osteocalcin). In the adenine-induced chronic renal failure (CRF) mouse VC model, PYP4 was found to obviously inhibit the aortic calcium level, and it also inhibited the serum creatinine, serum phosphorus and serum BUN levels. PYP4 (least molecular weight) showed the best inhibitory effect on calcification and may be considered as a candidate drug with therapeutic potential for inhibiting cellular damage and osteoblast differentiation induced by the HAP crystals.

Effects of phosphorus and calcium to phosphorus consumption ratio on mineral metabolism and cardiometabolic health.

Phosphorus is a common additive used in food processing that is typically consumed in excess of the recommended daily allowance; however, our knowledge of its effects on health, in the context of normal renal function, is limited. Unlike phosphorus, calcium intake is generally less than recommended, and it has been hypothesized that the calcium to phosphorus ratio may be partly responsible for the proposed negative health consequences. Therefore, this study sought to determine the effects of increased phosphorus additive intake, in the context of high calcium consumption, on endocrine markers of mineral metabolism and cardiometabolic health. An outpatient feeding study was performed in which healthy adults were fed a run-in control diet for 2 weeks followed by a phosphorus additive enhanced diet with supplemental calcium to an approximate ratio of 1 (experimental diet) for 2 weeks. Blood and urine samples were collected, and participants had brachial flow-mediated dilatation measured, with analyses comparing follow-up measures to baseline. Two weeks of experimental diet increased serum fibroblast growth factor 23 concentrations but lowered body weight and serum leptin; however, other phosphorus responsive factors such as osteopontin and osteocalcin did not increase. A complementary study in male mice also demonstrated that the regulation of known dietary phosphorus responsive factors was mostly abrogated when dietary calcium was raised in parallel with phosphorus. In conclusion, the study identifies weight, leptin and insulin as responsive to dietary phosphorus and that certain aspects of the systemic phosphorus response are attenuated by a corresponding high calcium intake.

The Solanum glaucophyllum Desf. extract reduces mineralized matrix synthesis in osteogenically differentiated rat mesenchymal stem cells in vitro.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 µl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 µl of extract/L containing 0.4 µg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 µg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 µg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.

Abnormal Lysosomal Positioning and Small Extracellular Vesicle Secretion in Arterial Stiffening and Calcification of Mice Lacking Mucolipin 1 Gene.

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1-/- mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1-/- mice as compared to their wild-type littermates. Besides, Mcoln1-/- mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1-/- mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1-/- mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.

Loss and rescue of osteocalcin and osteopontin modulate osteogenic and angiogenic features of mesenchymal stem/stromal cells.

Noncollagenous proteins in the bone extracellular matrix, such as osteocalcin (OC) and osteopontin (OPN), inherent to evolution of bone as a skeletal tissue, are known to regulate bone formation and mineralization. However, the fundamental basis of this regulatory role remains unknown. Here, for the first time, we use mouse mesenchymal stem/stromal cells (MSC) lacking both OC and OPN to investigate the mechanistic roles of OC and OPN on the proliferation capacity and differentiation ability of MSC. We found that the loss of OC and OPN reduces stem cells self-renewal potential and multipotency, affects their differentiation into an osteogenic lineage, and impairs their angiogenic potential while maintaining chondrogenic and adipogenic lineages. Moreover, loss of OC and OPN compromises the extracellular matrix integrity and maturation, observed by an unexpected enhancement of glycosaminoglycans content that are associated with a more primitive skeletal connective tissue, and by a delay on the maturation of mineral species produced. Interestingly, exogenously supplemented OC and OPN were able to rescue MSC proliferative and osteogenic potential along with matrix integrity and mineral quality. Taken together, these results highlight the key contributions of OC and OPN in enhancing osteogenesis and angiogenesis over primitive connective tissue, and support a potential therapeutic approach based on their exogenous supplementation.

The Antiosteoporosis Effects of Yishen Bugu Ye Based on Its Regulation on the Differentiation of Osteoblast and Osteoclast.

Yishen Bugu Ye (YSBGY), a traditional Chinese medicine comprising 12 types of medicinal herbs, is often prescribed in China to increase bone strength. In this study, the antiosteoporotic effects of YSBGY were investigated in C57BL/6 mice afflicted with dexamethasone- (Dex-) induced osteoporosis (OP). The results showed that YSBGY reduced the interstitial edema in the liver and kidney of mice with Dex-induced OP. It also increased the number of trabecular bone elements and chondrocytes in the femur, promoted cortical bone thickness and trabecular bone density, and modulated the OP-related indexes in the femur and tibia of OP mice. It also increased the serum concentrations of type I collagen, osteocalcin, osteopontin, bone morphogenetic protein-2, bone morphogenetic protein receptor type 2, C-terminal telopeptide of type I collagen, and runt-related transcription factor-2 and reduced those of tartrate-resistant acid phosphatase 5 and nuclear factor of activated T cells in these mice, suggesting that it improved osteoblast differentiation and suppressed osteoclast differentiation. The anti-inflammatory effect of YSBGY was confirmed by the increase in the serum concentrations of interleukin- (IL-) 33 and the decrease in concentrations of IL-1, IL-7, and tumor necrosis factor-α in OP mice. Furthermore, YSBGY enhanced the serum concentrations of superoxide dismutase and catalase in these mice, indicating that it also exerted antioxidative effects. This is the first study to confirm the antiosteoporotic effects of YSBGY in mice with Dex-induced OP, and it showed that these effects may be related to the YSBGY-induced modulation of the osteoblast/osteoclast balance and serum concentrations of inflammatory factors. These results provide experimental evidence supporting the use of YSBGY for supporting bone formation in the clinical setting.