Increase In Calcium Research Articles

Evaluation of Biological Mechanisms of Quanduzhong Capsule for Treating Osteoporosis by Integrating Untargeted Metabolomics and Network Pharmacology

AbstractOsteoporosis (OP) is a metabolic disease characterized by bone formation and resorption disturbances. Quanduzhong Capsule (QDZC) is a common treatment for OP in China; however, the effective components and metabolites of the drug after oral administration remain largely unknown. This study aims to identify the active components, analyze the metabolite changes, and investigate the underlying mechanism against OP. In the study, ovariectomy-induced rat OP model was established, then treated with QDZC. Alendronate sodium tablets (ASTs) were used as a reference drug. The chemical constituents of QDZC were analyzed by UPLC-QTOF-MS (ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry) and network pharmacology. The metabolomics was used to analyze differences in serum metabolites of rats in different groups [Sham, Model, Model + QDZC, and Model + AST] at 4, 8, and 12 weeks. Body weight and bone mineral density (BMD) were assessed. Enzyme-linked immunosorbent assay was used to determine serum levels of Akt, p-Akt, ERK, and p-ERK. Our data suggested 86 different chemicals from QDZC, including nine core compounds. QDZC significantly regulated 25 biomarkers linked to arachidonic acid metabolism and unsaturated fatty acid biosynthesis, and promoted serum expression of Akt, p-Akt, ERK, and p-ERK. QDZC might act by activating PI3K-Akt and MAPK signaling pathways. In addition, QDZC may use arachidonic acid derivatives to inhibit osteoclast generation and bone resorption and enhance calcitriol formation to improve calcium absorption and increase bone mass.

Development and characterization of an optimized novel drink from three varieties of sprouted quinoa

Sprouted products are a food alternative that contribute to improving human nutrition. Quinoa has a large amount of natural antioxidants. The dry and hard grains when they undergo a germination process change to tender and slightly acidic sprouts that contain amino acids, minerals, vitamins, enzymes that help digestion and antioxidants. Furthermore, sprouted quinoa has a high protein content and a low concentration of saponins. Previous studies have shown that sprouted quinoa has shown an increase in protein, calcium and ascorbic acid content. Quinoa sprouts improve the organoleptic characteristics of quinoa grains; few studies have used sprouted quinoa in the preparation of beverages. The objective of this study was to prepare a beverage based on sprouted quinoa using a simplex lattice mix design to determine the optimal formula. Three varieties of sprouted quinoa were used: white (Salcedo INIA), red (INIA-415 Pasankalla), and black (INIA 420-Negra Collana). The quinoa was incubated at 30°C in the dark for 48 hours at a relative humidity of 98%. Ten treatments with different proportions of the three types of sprouted quinoa were used. The antioxidant capacity and consumer satisfaction of the ten treatments were evaluated. The ABTS method was used to measure the antioxidant capacity of each treatment. The optimal drink mix was composed of 81.67% sprouted black quinoa, 18.33% sprouted white quinoa, and 0% sprouted red quinoa. Furthermore, the consumer acceptability results were similar across all treatments (p>0.05). A nutritional analysis revealed significant differences (p<0.05) between the optimized drink and the control treatment. The microbiological analysis demonstrated that the formulated drink met the required sanitary norms for aerobic mesophiles, coliforms, molds, yeasts, and absence of Salmonella. The quinoa beverage made from three varieties of germinated quinoa represents a novel alternative to the current drinkable products marketed in Peru due to its nutritional content, antioxidants, and ease of preparation. Key words: Sprouts, Chenopodium quinoa, ABTS, Design-expert, Mixture design, antioxidant capacity, acceptability

Abaloparatide Maintains Normal Rat Blood Calcium Level in Part Via 1,25-Dihydroxyvitamin D/osteocalcin Signaling Pathway.

The PTH-related peptide(1-34) analog, abaloparatide (ABL), is the second anabolic drug available for the treatment of osteoporosis. Previous research demonstrated that ABL had a potent anabolic effect but caused hypercalcemia at a significantly lower rate. However, the mechanism by which ABL maintains the stability of blood calcium levels remains poorly understood. Our in vivo data showed that ABL treatment (40 µg/kg/day for 7 days) significantly increased rat blood level of 1,25-dihydroxyvitamin D [1,25-(OH)2D] without raising the blood calcium value. ABL also significantly augmented the carboxylated osteocalcin (Gla-Ocn) in the blood and bone that is synthesized by osteoblasts, and increased noncarboxylated Ocn, which is released from the bone matrix to the circulation because of osteoclast activation. The in vitro data showed that ABL (10 nM for 24 hours) had little direct effects on 1,25-(OH)2D synthesis and Gla-Ocn formation in nonrenal cells (rat osteoblast-like cells). However, ABL significantly promoted both 1,25-(OH)2D and Gla-Ocn formation when 25-hydroxyvitamin D, the substrate of 1α-hydroxylase, was added to the cells. Thus, the increased 1,25-(OH)2D levels in rats treated by ABL result in high levels of Gla-Ocn and transient calcium increase in the circulation. Gla-Ocn then mediates calcium ions in the extracellular fluid at bone sites to bind to hydroxyapatite at bone surfaces. This regulation by Gla-Ocn at least, in part, maintains the stability of blood calcium levels during ABL treatment. We conclude that the signaling pathway of ABL/1,25-(OH)2D/Gla-Ocn contributes to calcium homeostasis and may help understand the mechanism of ABL for osteoporosis therapy.

Assessing the efficacy of ameliorants on saline-sodic soils: Laboratory insights for reclamation strategies

This study presents the combined findings of laboratory experiments conducted to assess the efficacy of various ameliorants on saline-sodic soils in the foothill plain of Ile Alatau in the Northern Tianshan region. The investigation focused on the influence of phosphogypsum, elemental sulfur, nano sulfur, and sulfuric acid on the ionic composition of the soil solution and their impact on the soil-absorbing complex. Different doses of these ameliorants were applied to saline-sodic soil samples, and their incubation period was observed. The analysis of the aqueous extract of the soil emphasized the presence of bicarbonate, carbonate, sulfate, calcium, and sodium ions. The results revealed that sulfuric acid was the most effective ameliorant, rapidly neutralizing extreme alkalinity, reducing bicarbonate and carbonate ion content, and increasing sulfate and sodium ion concentrations. Elemental sulfur ranked second in effectiveness, significantly decreasing bicarbonate and carbonate ions and increasing sulfate and sodium ions. Phosphogypsum exhibited the lowest effectiveness, causing reductions in bicarbonate and carbonate ions and modest increases in sulfate and calcium ions. The study demonstrated that the introduction of phosphogypsum led to an increase in calcium and sulfate ions in the soil solution, while elemental sulfur and sulfuric acid significantly increased the sulfate ion content. Sulfuric acid exhibited the highest efficacy among the ameliorants, completely neutralizing normal carbonates and reducing alkalinity in the soil solution. The formation of subsoil gypsum through the interaction of sulfuric acid with calcium carbonates facilitated the displacement of sodium from the soil-absorbing complex. These findings contribute to our understanding of the processes involved in the amelioration of saline-sodic soils and provide insights into effective soil management practices. They serve as a theoretical basis for developing strategies for the reclamation of such soils worldwide. The research highlights sulfuric acid as the most effective ameliorant for saline-sodic soils, resulting in a significant rearrangement of the soil's ionic composition. Further research and field studies are necessary to validate and refine these laboratory findings for practical applications in soil improvement methods.

Calcium dynamics and associated patterns of milk constituents in early lactation multiparous Holsteins

Immediately following parturition, physiological demands for calcium increase substantially to support the onset of milk production. As a result, transitioning dairy cows may experi­ence subclinical hypocalcemia (SCH) at some point during early lactation. Cows respond to this homeostatic challenge dynamically, such that serum total calcium concentrations (tCa) at 1 and 4 days in milk (DIM) can be used to classify cows into 4 calcium dynamic groups: normocalcemic (NC: SCH at no DIM), transient (tSCH: SCH at 1 DIM only), delayed (dSCH: SCH at 4 DIM only) and persistent (pSCH: SCH at both 1 and 4 DIM). Identifying cows in the pSCH and dSCH groups is important clinically because these groups are at greater risk of adverse health events compared to the NC and tSCH cows. Our objective was to describe the temporal patterns of milk constituents for cows in each calcium group using Fourier-transform infrared (FTIR) spectroscopic analysis of milk samples. Additionally, we hoped to ascertain whether these methods might be valuable as a diagnostic tool for grouping cows based on calcium dynamics at a clinically useful timepoint.

SKORING CORONARY ARTERY CALSIUM PADA INDIVIDU USIA DEWASA AKHIR DAN LANSIA DENGAN HIPERTENSI

Hypertension causes an increase in coronary artery calcium (CAC), which is a strong predictor of new-onset hypertension and occurrences related to atherosclerotic cardiovascular disease in people either with or without hypertension. The medical application of CAC scoring to adjust hypertension management has received slight attention and is poorly defined. In Indonesia, research on the benefits of CAC assessment for hypertension is yet very limited. This study will show the association of hypertension with CAC rating, age, and gender. This cross-sectional study used secondary data in the form of medical records from 163 patients (88 men, 75 women; range 40-70 years) at Siloam Hospital TB Simatupang. Sample collection was carried out using consecutive nonrandom sampling. Only medical records with complete physical examination results and CAC scoring will be further analyzed. The Mann-Whitney statistical test was carried out using the SPSS 25.0 program to see the relationship between CAC scoring and hypertension, with a value of p=0.002 and a significance level of p<0.05. To assess the relationship between age and gender with the incidence of hypertension, a chi square test was carried out, with each value p = 0.011 and p = 0.141 (p <0.05). The results showed a relationship between CAC scoring and hypertension in late adulthood and elderly individuals.

Modification mechanism of flue gas desulfurization gypsum on fly ash and ground granulated blast-furnace slag alkali-activated materials: Promoting green cementitious material

Alkali activation and the combination of fly ash (FA) and ground granulated blast-furnace slag (GGBS) has been used for cementitious material, which is seen as an effective method to dispose of industrial solid waste. The GGBS-FA AAMs have the characteristic of drying shrinkage and a large number of tiny pores distributed in the matrix, resulting in strength deterioration. The paper is aimed to optimize the strength property by incorporating another solid waste of flue gas desulfurization gypsum (DG), which could be regarded as a ternary alkali-activated materials (AAMs). The unconfined compression tests are carried out to study the effects of DG content on strength with different curing times, and the effects from the modulus of the alkali activator are also analyzed. Then X-ray diffraction (XRD) and Scanning Electron Microscope (SEM) are performed to reveal the microscopic mechanism of the effects of DG on the mechanical property. Results show that the incorporation of DG can significantly improve the early strength due to the increase of calcium. The SO42- in DG continuously reacts with Ca2+ and active silicate to form ettringite during the curing period, which can fill micro-pores and tiny cracks to form a denser matrix structure. In addition, the presence of SO42- has the effect of secondary enhanced activation, further promoting the polymerization reaction and enhancing the strength, especially for the low alkaline reaction system. These all contribute to the strength improvement. It should be noted that the DG content must be controlled to prevent the volume-filling effect that leads the AAMs matrix to be loose and porous, and the generation of geopolymer gel would also be impeded in a high-calcium reaction system, further influencing the final strength. The results will promote the application of high-performance, cost-saving, and green low-carbon cementitious material based on the synergistic utilization of GGBS, FA and DG.

Electron Leak From the Mitochondrial Electron Transport Chain Complex I at Site IQ Is Crucial for Oxygen Sensing in Rabbit and Human Ductus Arteriosus.

Background As partial pressure of oxygen (pO2) rises with the first breath, the ductus arteriosus (DA) constricts, diverting blood flow to the pulmonary circulation. The DA's O2 sensor resides within smooth muscle cells. The DA smooth muscle cells' mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) in proportion tooxygen tension, causing vasoconstriction by regulating redox-sensitive ion channels and enzymes. To identify which ETC complex contributes most to DA O2 sensing and determine whether ROS mediate O2 sensing independent of metabolism, we used electron leak suppressors, S1QEL (suppressor of site IQ electron leak) and S3QEL (suppressor of site IIIQo electron leak), which decrease ROS production by inhibiting electron leak from quinone sites IQ and IIIQo, respectively. Methods and Results The effects of S1QEL, S3QEL, and ETC inhibitors (rotenone and antimycin A) on DA tone, mitochondrial metabolism, O2-induced changes in intracellular calcium, and ROS were studied in rabbit DA rings, and human and rabbit DA smooth muscle cells. S1QEL's effects on DA patency were assessed in rabbit kits, using micro computed tomography. In DA rings, S1QEL, but not S3QEL, reversed O2-induced constriction (P=0.0034) without reducing phenylephrine-induced constriction. S1QEL did not inhibit mitochondrial metabolism or ETC-I activity. In human DA smooth muscle cells, S1QEL and rotenone inhibited O2-induced increases in intracellular calcium (P=0.02 and 0.001, respectively), a surrogate for DA constriction. S1QEL inhibited O2-induced ROS generation (P=0.02). In vivo, S1QEL prevented O2-induced DA closure (P<0.0001). Conclusions S1QEL, but not S3QEL, inhibited O2-induced rises in ROS and DA constriction ex vivo and in vivo. DA O2 sensing relies on pO2-dependent changes in electron leak at site IQ in ETC-I, independent of metabolism. S1QEL offers a therapeutic means to maintain DA patency.

The Effect Of High-Intensity Exercise Training On Serum Calcium Of Female Combat Athletes

Purpose: Cell homeostasis is an essential factor in maintaining cellular health and intracellular mechanisms, which can also be associated with inflammatory factors and increased stress for muscle cell health. Calcium is also a necessary factor in cell health and is to establish homeostasis in the body. This study aimed to investigate the effect of high-intensity training on serum calcium levels in combat women. Method: For this purpose, 18 women karateka in the age range of 17 to 23 years were randomly divided into 2 groups: control (n=7) and exercise ((n=11). The exercise group performed proper exercises to improve karate and aerobic techniques for 6 weeks in the intensities of more than 80% of the maximum heart rate reserve. Physiological indices and blood samples were measured 24 hours before and 48 hours after the training protocol. To measure calcium cell biomarker, the Pars Azmoon calcium kit was used. Research data were analysed by covariance test at the significant level (P≤0.05) using SPSS software version 25. Results: After six weeks of high-intensity training, there was a significant difference in serum calcium levels in the exercise group compared to the control group (P = 0.002). Also, the results within the group showed a significant increase in serum calcium (P = 0.016) in the exercise group compared to baseline conditions. Conclusions: High intensity training seems to increase serum calcium in female athletes, and this can lead to precursors to cellular disorders, muscle damage and increase inflammatory factors and ultimately decrease the performance of female athletes. However, more research is essential in this regard.

Isolation and Biological Activity of Iezoside and Iezoside B, SERCA Inhibitors from Floridian Marine Cyanobacteria.

Marine cyanobacteria are a rich source of bioactive natural products. Here, we report the isolation and structure elucidation of the previously reported iezoside (1) and its C-31 O-demethyl analogue, iezoside B (2), from a cyanobacterial assemblage collected at Loggerhead Key in the Dry Tortugas, Florida. The two compounds have a unique skeleton comprised of a peptide, a polyketide and a modified sugar unit. The compounds were tested for cytotoxicity and effects on intracellular calcium. Both compounds exhibited cytotoxic activity with an IC50 of 1.5 and 3.0 μΜ, respectively, against A549 lung carcinoma epithelial cells and 1.0 and 2.4 μΜ against HeLa cervical cancer cells, respectively. In the same cell lines, compounds 1 and 2 show an increase in cytosolic calcium with approximate EC50 values of 0.3 and 0.6 μΜ in A549 cells and 0.1 and 0.5 μΜ, respectively, in HeLa cells, near the IC50 for cell viability, suggesting that the increase in cytosolic calcium is functionally related to the cytotoxicity of the compounds and consistent with their activity as SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) inhibitors. The structure-activity relationship provides evidence that structural changes in the sugar unit may be tolerated, and the activity is tunable. This finding has implications for future analogue synthesis and target interaction studies.

P-096 Prediction of fertilization success by voltage-dependent intracellular Ca2+ dynamics in non-normozoospermic human semen samples undergoing in vitro fertilization: preliminary results

Abstract Study question Does the change in intracellular calcium levels evoked by artificial membrane depolarizations predict fertilization success in a non-normozoospermic semen sample undergoing assisted reproductive techniques? Summary answer The increase in the intracellular calcium after artificial plasma membrane depolarization correlates negatively with fertilization rates in conventional IVF but not with ICSI What is known already Sperm capacitation-related features, like the regulation of the intracellular calcium concentration ([Ca2+]i) and the hyperpolarization of the plasma membrane potential (Vm), have been proposed as predictors of success during artificial reproduction techniques (ART). Intriguingly, upon Vm depolarization, sperm exhibit a rapid and transitory increase in the [Ca2+]i controlled, likely, by the activation of the sperm-specific calcium channel CatSper. The regulation of CatSper activity is essential for sperm function and is crucial for fertilization. Therefore we wondered if the Vm depolarization-dependent rise in the [Ca2+]i can be used to predict fertilization success rates in non-normozoospermic semen samples of men undergoing ART. Study design, size, duration This prospective study employed unidentified semen samples from 29 non-normozoospermic men (37.8 ± 5.6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were analyzed as a control. The depolarization-dependent [Ca2+]i changes were evaluated by flow cytometry under four different Vm conditions: resting (variable among samples), –64, –46, and –32 mV. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality, Ca2+, and Vm-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. The kinetical analysis of the transitory [Ca2+]i increase as well as the statistical comparisons were performed using custom-made R programming language code. Main results and the role of chance Analysis of resting Vm showed that normozoospermic semen samples presented more depolarized Vm than non-normozoospermic controls (–75.7 ± 6.9 vs –67.3 ± 10.5 mV, respectively, p = 0.02). The former group of semen samples was either used for conventional IVF (n = 11) or ICSI (n = 14). The semen samples in these last categories showed a tendency to present a more depolarized Vm than non-normozoospermic samples (–69.9 ± 8.8 and –66.6 ± 11.3 mV, respectively). No differences were seen in the resting Ca2+ levels. We then induced a Vm hyperpolarization by adding the K+ ionophore, valinomycin followed by increasing concentrations of extracellular K+. Each depolarizing stimulus evoked a fast and transitory increase in the [Ca2+]i. We then calculated seven different kinetic parameters of such responses: basal Ca2+ level, increase and decrease velocity, maximum Ca2+ reached, response durability, and area under the curve (AUC). The basal Ca2+ level reached after depolarizing stimuli correlated negatively (R = –0.64, p = 0.03) with the conventional IVF fertilization ratio (number of zygotes/total number of mature oocytes) when the Vm was clamped either to –64 and –46 mV. A negative and significant correlation (R = –0.76, p = 0.006) was seen for the AUC analysis at –64 mV. No relevant correlations were found in the ICSI samples. Limitations, reasons for caution This is an in vitro study, and caution must be taken when extrapolating these results in vivo. The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). Wider implications of the findings Further research into the molecular regulation of the CatSper channel could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. This protocol is fast and simple to perform in a laboratory equipped with a flow cytometer or any fluorescence-based reader. Trial registration number NA

Laboratory experiments and modelling of the geochemical interaction of a gabbro-anorthosite with seawater and supercritical CO2: A mineral carbonation study

The potential for mineral carbonation of carbon dioxide (CO2) in a plutonic mafic rock is addressed in this work through a set of laboratory experiments on a gabbro-anorthosite sample from the Torrão - Odivelas Massif (Portugal). The experiment was conducted in two stages under pressure (8 MPa) and temperature (313.15 K) conditions similar to those expected around a CO2 injection well. Stage-I simulated dissolution conditions, with a crushed rock sample (1–3 mm) exposed for 30 days to CO2 supersaturated seawater (0.576 M). Stage-II favoured carbonation conditions; cubic rock specimens (8 cm3) were brought in contact with the Stage-I seawater subsaturated in CO2 (0.095 M).A multi-analytical methodology was applied to follow the mineralogical and geochemical evolution of rock and seawater during both stages. At the end of Stage-I, the aqueous phase showed a marked increase in iron (Fe), magnesium (Mg) and calcium (Ca) ions and milder increase in silicon (Si) and aluminium (Al) ions. At the end of Stage-II, a decrease in Si and Al concentrations was observed while the Fe, Mg, Ca concentrations still increased, albeit at a lower rate. No significant geochemical variations were detected in the bulk solid phase. The bulk X-ray diffraction (XRD) analyses detected halite as a new mineral phase, while the grazing-XRD design revealed the presence of dolomite. Moreover, the elemental distribution mapping demonstrated the spatial association of carbon with magnesium indicating magnesite crystallisation. The geochemical model developed in CrunchFlow code was able to reproduce the experimental observations and simulate the chemical behaviour for longer time periods.Overall, the experiments and model point towards a mineral carbonation potential in the Torrão - Odivelas Massif, with further studies being required to upscale the laboratory results to field scale.

Inverse Regulation of Cartilage Neogenesis at Physiologically Relevant Calcium Conditions by Human Articular Chondrocytes and Mesenchymal Stromal Cells.

Elaborate bioreactor cultivation or expensive growth factor supplementation can enhance extracellular matrix production in engineered neocartilage to provide sufficient mechanical resistance. We here investigated whether raising extracellular calcium levels in chondrogenic cultures to physiologically relevant levels would provide a simple and inexpensive alternative to enhance cartilage neogenesis from human articular chondrocytes (AC) or bone marrow-derived mesenchymal stromal cells (BMSC). Interestingly, AC and BMSC-derived chondrocytes showed an opposite response to a calcium increase from 1.8 mM to 8 mM by which glycosaminoglycan (GAG) and collagen type II production were elevated during BMSC chondrogenesis but depressed in AC, leading to two-fold higher GAG/DNA values in BMSC-based neocartilage compared to the AC group. According to control treatments with Mg2+ or sucrose, these effects were specific for CaCl2 rather than divalent cations or osmolarity. Importantly, undesired pro-hypertrophic traits were not stimulated by calcium treatment. Specific induction of PTHrP mRNA and protein by 8.0mM calcium only in AC, along with negative effects of recombinant PTHrP1-34 on cartilage matrix production, suggested that the PTHrP pathway contributed to the detrimental effects in AC-based neocartilage. Altogether, raising extracellular calcium levels was discovered as a novel, simple and inexpensive stimulator for BMSC-based cartilage neogenesis without the need for special bioreactors, whereas such conditions should be avoided for AC.

Feasibility of laser-induced breakdown spectroscopy to elucidate elemental changes in human tooth ankylosis

Tooth ankylosis is a pathological condition associated with the loss of physiological tooth mobility when the firm fusion between the alveolar bone and the tooth root occurs. Causes of dental ankylosis are uncertain, so the analysis of elemental distribution in ankylotic and surrounding tissues could provide additional information about its initiation and progression. Here, we used Laser-Induced Breakdown Spectroscopy (LIBS) to determine differences in the mineral composition among ankylotic tissue, bone, and dental tissue taking advantage of its high throughput and multi-elemental capability. Elemental imaging was performed with a spatial resolution of 30 μm to evaluate the distribution of carbon, calcium, magnesium, phosphorus, and strontium in human tooth. To further verify the difference in the mineral composition of ankylotic tissue, the semi-quantitative content of these elements was compared within the region of interest. We revealed a significant increase in calcium, magnesium, and phosphorus contents in the ankylotic tissues. However, the contents of magnesium and phosphorus were not significantly changed. This finding suggests a mineral disbalance only of just certain elements in the tooth-bone interface area during the spread of ankylosis associated with an intense calcification of connective tissue. This paper brings a feasibility study and shows the way of LIBS data interpretation. We propose that the LIBS analysis on a micro-scale can contribute to the understanding of ankylotic tissue composition and can distinguish even small differences of carbon, calcium, magnesium, phosphorus, and strontium contents on the tooth-bone boundary during the initiation of ankylosis. Therefore, it represents a new useful tool for their future, more extensive analyses.

Cold atmospheric nitrogen plasma induces metal-initiated cell death by cell membrane rupture and mitochondrial perturbation.

Cold atmospheric plasma (CAP) is a novel biomedical tool used for cancer therapy. A device using nitrogen gas (N2 CAP) produced CAP that induced cell death through the production of reactive nitrogen species and an increase in intracellular calcium. In this study, we investigated the effect of N2 CAP-irradiation on cell membrane and mitochondrial function in human embryonic kidney cell line 293T. We investigated whether iron is involved in N2 CAP-induced cell death, as deferoxamine methanesulfonate (an iron chelator) inhibits this process. We found that N2 CAP induced cell membrane disturbance and loss of mitochondrial membrane potential in an irradiation time-dependent manner. BAPTA-AM, a cell-permeable calcium chelator, inhibited N2 CAP-induced loss of mitochondrial membrane potential. These results suggest that disruption of intracellular metal homeostasis was involved in N2 CAP-induced cell membrane rupture and mitochondrial dysfunction. Moreover, N2 CAP irradiation generated a time-dependent production of peroxynitrite. However, lipid-derived radicals are unrelated to N2 CAP-induced cell death. Generally, N2 CAP-induced cell death is driven by the complex interaction between metal movement and reactive oxygen and nitrogen species produced by N2 CAP.

#4408 ASSOCIATION BETWEEN CKD-MBD AND SYMPTOM BURDEN IN OLDER PATIENTS WITH ADVANCED CKD – RESULTS FROM THE EQUAL STUDY

Abstract Background and Aims Patients with advanced chronic kidney disease (CKD) typically develop a constellation of various non-specific symptoms known as the uremic syndrome. This symptom burden affects patients’ quality of life and is one of the most relevant reasons for initiating dialysis. Several factors are thought to be involved in symptoms development, including mineral bone disorder (CKD-MBD). However, evidence linking deranged mineral biomarkers and CKD symptoms is mostly limited to few symptoms in dialysis patients. We studied the association between PTH, phosphate and calcium and the overall symptom burden in a cohort of elderly non-dialysis patients with advanced CKD, during 5 years of follow up. Secondly, we investigated whether these biomarkers are specifically associated with a wide spectrum of CKD-related signs and symptoms. Method We used data from the EQUAL study, which includes patients aged ≥65 with eGFR ≤20 ml/min/1.73m2 from six European countries. Symptoms were repeatedly assessed at 6-month intervals through a questionnaire on 33 CKD-related signs and symptoms. Generalized linear mixed models were used to study the association between mineral biomarkers and the overall symptom number (range 0–33), the overall symptom severity (range 0–165, with higher scores indicating higher severity), the probability of having each symptom and its severity (range 1–5). Models were adjusted for age, sex, Charlson Comorbidity Index, kidney function, albumin, hemoglobin and CKD-MBD medications. Results At baseline, the 1396 patients included in the study had a mean (SD) of 12.6 (±6.4) symptoms and a median symptom severity score of 32 [19-50], with the most prevalent symptoms being fatigue, loss of strength, muscle cramps, bone pain, swelling in legs, dry skin, and sexual disorders. Among the mineral biomarkers, only PTH had a significant association with the overall symptom number and severity. Furthermore, a doubling in PTH was associated with an increased probability of reporting shortness of breath (OR 1.13 [1.01, 1.27], p 0.03) and decreased probability of loss of strength (OR 0.90 [0.81, 1.00], p 0.05). Both PTH and phosphate had significant U-shaped associations with gastrointestinal symptoms (decreased appetite, nausea, and vomiting). A doubling in phosphate level was linearly associated to the severity of muscle cramps (β 0.20 [0.06, 0.36], p 0.007) and bone pain (β 0.24 [0.09, 0.39], p 0.002), in patients who experienced these symptoms. Phosphate was also non-linearly associated with the probability of having trouble falling asleep. One mmol/L increase in calcium was associated with both a decreased probability of having muscle cramps (OR 0.50 [0.25, 0.98], p 0.04) and its severity (β -0.43 [-0.69, -0.17], p 0.001). Conclusion Serum levels of PTH are associated with the overall symptom burden in older non-dialysis patients with advanced CKD, and PTH, phosphate, and calcium are specifically and independently associated with some of the individual symptoms in this population.

Pre-flight body weight effects on urinary calcium excretion in space

Microgravity-induced bone loss increases urinary calcium excretion which increases kidney stone formation risk. Not all individuals show the same degree of increase in urinary calcium and some pre-flight characteristics may help identify individuals who may benefit from in-flight monitoring. In weightlessness the bone is unloaded, and the effect of this unloading may be greater for those who weigh more. We studied whether pre-flight body weight was associated with increased in-flight urinary calcium excretion using data from Skylab and the International Space Station (ISS). The study was reviewed and approved by the National Aeronautics and Space Administration (NASA) electronic Institutional Review Board (eIRB) and data were sourced from the Longitudinal Study of Astronaut Health (LSAH) database. The combined Skylab and ISS data included 45 participants (9 Skylab, 36 ISS). Both weight and day in flight were positively related to urinary calcium excretion. There was also an interaction between weight and day in flight with higher weight associated with higher calcium excretion earlier in the mission. This study shows that pre-flight weight is also a factor and could be included in the risk assessments for bone loss and kidney stone formation in space.

#3769 MECHANOSENSITIVE ION CHANNEL PIEZO1 MEDIATES BLOOD PRESSURE HOMEOSTASIS BY REGULATING RENIN EXPRESSION IN MICE

Abstract Background and Aims The renin-angiotensin system (RAS) plays a crucial role in the maintenance of fluid-electrolyte and blood pressure homeostasis. Renin, the step-limiting protease of the RAS, is produced, stored, and secreted by the renal juxtaglomerular (JG) cells, which are closely regulated by the blood pressure. Studies have shown that local blood flow-associated shear stress play an important role in renin expression and secretion. To date, however, the molecular identity through which JG cells respond to the blood pressure remains unclear. Method In this study, we determined the expression and subcellular localization of Piezo1 in JG cells by qPCR, Western blot, Immunofluorescence (IF), and multiplex immunohistochemistry. Then, a Fluo-4 AM-based calcium-imaging system was used to detect the dynamic changes of intracellular calcium in response to Piezo1 specific agonist Yoda1 and mechanical stress (MS)-induced by perfusion. Next, CRISPR/Cas9 technology was used to generate Piezo1 knockout JG cells to further confirm its contribution. Then, we used RNA-seq experiment to investigate the downstream signaling of Piezo1 in JG cells. Finally, we generated adeno-associated virus (AAV)-mediated kidney-specific Piezo1 knockdown mice to investigate the in vivo effect of Piezo1. Results We found that the mechanosensitive ion channel Piezo1 was co-expressed with renin in JG cells in mouse kidney slides as well as As4.1 cells, a commonly used JG cell line. Activation of Piezo1 by its agonist Yoda1 induced an intracellular calcium increase and reduced the expression of renin in these cells, while knockout of Piezo1 in JG cells abolished the effect of Yoda1. Meanwhile, fluid induced mechanical stress also led to an intracellular calcium increase and renin downregulation in wildtype but not Piezo1 knockout JG cells. Mechanistically, we demonstrated that activation of Piezo1 upregulated the Ptgs2 expression via the calcineurin-NFAT pathway and increased the production of Ptgs2 downstream molecule COX-2 and PGE2 in JG cells, which inhibited cAMP production and reduced renin expression in JG cells. In mice, we demonstrated that activation of Piezo1 significantly downregulated the blood pressure in wildtype but not kidney-specific Piezo1 knockdown mice. Conclusion In summary, these results revealed that activation of Piezo1 could lead to downregulation of the renin expression in vitro and in vivo, subsequently reduction of blood pressure, highlighting its therapeutic potential as a drug target of the renin-angiotensin system.

Impact of cryopreservation on viability, gene expression and function of enteric nervous system derived neurospheres.

Introduction: Impairment of both the central and peripheral nervous system is a major cause of mortality and disability. It varies from an affection of the brain to various types of enteric dysganglionosis. Congenital enteric dysganglionosis is characterized by the local absence of intrinsic innervation due to deficits in either migration, proliferation or differentiation of neural stem cells. Despite surgery, children's quality of life is reduced. Neural stem cell transplantation seems a promising therapeutic approach, requiring huge amounts of cells and multiple approaches to fully colonize the diseased areas completely. A combination of successful expansion and storage of neural stem cells is needed until a sufficient amount of cells is generated. This must be combined with suitable cell transplantation strategies, that cover all the area affected. Cryopreservation provides the possibility to store cells for long time, unfortunately with side effects, i.e., upon vitality. Methods: In this study we investigate the impact of different freezing and thawing protocols (M1-M4) upon enteric neural stem cell survival, protein and gene expression, and cell function. Results: Freezing enteric nervous system derived neurospheres (ENSdN) following slow-freezing protocols (M1-3) resulted in higher survival rates than flash-freezing (M4). RNA expression profiles were least affected by freezing protocols M1/2, whereas the protein expression of ENSdN remained unchanged after treatment with protocol M1 only. Cells treated with the most promising freezing protocol (M1, slow freezing in fetal calf serum plus 10% DMSO) were subsequently investigated using single-cell calcium imaging. Freezing of ENSdN did not alter the increase in intracellular calcium in response to a specific set of stimuli. Single cells could be assigned to functional subgroups according to response patterns and a significant shift towards cells responding to nicotine was observed after freezing. Discussion: The results demonstrate that cryopreservation of ENSdN is possible with reduced viability, only slight changes in protein/gene expression patterns and without an impact on the neuronal function of different enteric nervous system cell subtypes, with the exception of a subtle upregulation of cells expressing nicotinergic acetylcholine receptors. In summary, cryopreservation presents a good method to store sufficient amounts of enteric neural stem cells without neuronal impairment, in order to enable subsequent transplantation of cells into compromised tissues.

Butyrate inhibits LPC-induced endothelial dysfunction by regulating nNOS-produced NO and ROS production

Lipids oxidation is a key risk factor for cardiovascular diseases. Lysophosphatidylcholine (LPC), the major component of oxidized LDL, is an important triggering agent for endothelial dysfunction and atherogenesis. Sodium butyrate, a short-chain fatty acid, has demonstrated atheroprotective properties. So, we evaluate the role of butyrate in LPC-induced endothelial dysfunction. Vascular response to phenylephrine (Phe) and acetylcholine (Ach) was performed in aortic rings from male mice (C57BL/6J). The aortic rings were incubated with LPC (10 μM) and butyrate (0.01 or 0.1 Mm), with or without TRIM (an nNOS inhibitor). Endothelial cells (EA.hy296) were incubated with LPC and butyrate to evaluate nitric oxide (NO) and reactive oxygen species (ROS) production, calcium influx, and the expression of total and phosphorylated nNOS and ERK½. We found that butyrate inhibited LPC-induced endothelial dysfunction by improving nNOS activity in aortic rings. In endothelial cells, butyrate reduced ROS production and increased nNOS-related NO release, by improving nNOS activation (phosphorylation at Ser1412). Additionally, butyrate prevented the increase in cytosolic calcium and inhibited ERk½ activation by LPC. In conclusion, butyrate inhibited LPC-induced vascular dysfunction by increasing nNOS-derived NO and reducing ROS production. Butyrate restored nNOS activation, which was associated with calcium handling normalization and reduction of ERK½ activation.