Expression Of TRPV6 Research Articles

The endocannabinoid system in bovine tissues: characterization of transcript abundance in the growing Holstein steer

BackgroundThe endocannabinoid system (ECS) is highly integrated with seemingly all physiological and pathophysiological processes in the body. There is increasing interest in utilizing bioactive plant compounds, for promoting health and improving production in livestock. Given the established interaction between phytochemicals and the ECS, there are many opportunities for identification and development of therapies to address a range of diseases and disorders. However, the ECS has not been thoroughly characterized in cattle, especially in the gastrointestinal tract. The objective of this study was to characterize the distribution and transcriptional abundance of genes associated with the endocannabinoid system in bovine tissues.MethodsTissues including brain, spleen, thyroid, lung, liver, kidney, mesenteric vein, tongue, sublingual mucosa, rumen, omasum, duodenum, jejunum, ileum and colon were collected from 10-mo old Holstein steers (n = 6). Total RNA was extracted and gene expression was measured using absolute quantification real time qPCR. Gene expression of endocannabinoid receptors CNR1 and CNR2, synthesis enzymes DAGLA, DAGLB and NAPEPLD, degradation enzymes MGLL and FAAH, and transient receptor potential vanilloids TRPV3 and TRPV6 was measured. Data were analyzed in R using a Kruskal-Wallis followed by a Wilcoxon rank-sum test. Results are reported as the median copy number/20 ng of equivalent cDNA (CN) with interquartile range (IQR).ResultsThe greatest expression of CNR1 and CNR2 was in the brain and spleen, respectively. Expression of either receptor was not detected in any gastrointestinal tissues, however there was a tendency (P = 0.095) for CNR2 to be expressed above background in rumen. Expression of endocannabinoid synthesis and degradation enzymes varied greatly across tissues. Brain tissue had the greatest DAGLA expression at 641 CN (IQR 52; P ≤ 0.05). DAGLB was detected in all tissues, with brain and spleen having the greatest expression (P ≤ 0.05). Expression of NAPEPLD in the gastrointestinal tract was lowest in tongue and sublingual mucosal. There was no difference in expression of NAPEPLD between hindgut tissues, however these tissues collectively had 592% greater expression than rumen and omasum (P ≤ 0.05). While MGLL was found to be expressed in all tissues, expression of FAAH was only above the limit of detection in brain, liver, kidney, jejunum and ileum. TRPV3 was expressed above background in tongue, rumen, omasum and colon. Although not different from each other, thyroid and duodenum had the greatest expression of TRPV6, with 285 (IQR 164) and 563 (IQR 467) CN compared to all other tissues (P < 0.05).ConclusionsThese data demonstrate the complex distribution and variation of the ECS in bovine tissues. Expression patterns suggest that regulatory functions of this system are tissue dependent, providing initial insight into potential target tissues for manipulation of the ECS.

98 Differential gene expression of the endocannabinoid system in bovine tissues

Abstract Because of the association with intake and metabolism, there is interest in the endocannabinoid system and how it may be manipulated to benefit livestock. With the recent interest in using industrial hemp as a feedstuff in ruminants, understanding the interaction between phytocannabinoids and the endocannabinoid system is necessary. However, the endocannabinoid system has not been specifically characterized in cattle, especially the gastrointestinal tract (GIT). The objective of this study was to characterize the distribution and transcriptional abundance of genes associated with the endocannabinoid system in cattle. Tissues including brain, spleen, thyroid, lung, liver, kidney, mesenteric vein, tongue, sublingual mucosa, rumen, omasum, duodenum, jejunum, ileum and colon were collected from 10-mo old Holstein steers (n = 6). Total RNA was extracted, and gene expression was measured using absolute quantification real time qPCR. Gene expression of endocannabinoid receptors CNR1 and CNR2, synthesis enzymes DAGLA, DAGLB and NAPEPLD, degradation enzymes MGLL and FAAH, and transient receptor potential vanilloids TRPV3 and TRPV6 was measured. Data were analyzed in R using a Kruskal-Wallis followed by a Wilcoxon rank-sum test. Results are reported as the median copy number/20 ng of equivalent cDNA (CN) with interquartile range (IQR). The greatest expression of CNR1 and CNR2 was in the brain and spleen, respectively. Expression of either receptor was not detected in any GIT tissues; however, there was a tendency (P = 0.095) for CNR2 to be expressed above background in rumen. Expression of endocannabinoid synthesis and degradation enzymes varied greatly across tissues. Brain tissue had the greatest expression at 641 CN (IQR 52; P ≤ 0.05) of DAGLA; however, expression in liver and most of the GIT tissues was below the limit of detection. DAGLB was detected in all tissues, with brain and spleen having the greatest expression (P ≤ 0.05). Expression of NAPEPLD in the GIT was least in tongue and sublingual mucosal. There was no difference in expression between hindgut tissues; however, these tissues collectively had 592% greater expression than rumen and omasum (P ≤ 0.05). While MGLL was found to be expressed in all tissues, expression of FAAH was only above the limit of detection in brain, liver, kidney, jejunum and ileum. TRPV3 was expressed above background in tongue, rumen, omasum and colon, with rumen having the greatest expression (198 CN, IQR 13.3; P = 0.02). Although not difference from each other, thyroid and duodenum had the greatest expression of TRPV6, with 285 (IQR 164) and 563 (IQR 467) CN compared with all other tissues (P &amp;lt; 0.05). These data demonstrate the complex distribution of the endocannabinoid system in cattle. Expression levels suggest that regulatory functions of this system are tissue dependent, providing initial insight into potential target tissues for manipulation of the endocannabinoid system.

Chitosan encapsulation soy peptide–calcium promotes calcium absorption and bone health of rats fed a low calcium diet

Calcium deficiency is considered widespread globally, and discovering calcium with high bioavailability is one of the keys to solving calcium deficiency. This study prepared a calcium delivery system with chitosan and soy peptide and investigated its characteristics, ability to promote calcium absorption, and effects on bone health. The experiment results reveal that chitosan encapsulates soybean peptide–calcium (COS–SPs–Ca) with carboxyl oxygen atoms, amino nitrogen atoms, and phosphate ions, which has a slow-release effect in simulated gastrointestinal digestion. In calcium-deficient rats, COS–SPs–Ca has beneficial effects on serum biochemical parameters, bone biomechanics, and bone microarchitecture, and significantly up-regulated the gene expression of TRPV5, TRPV6, PepT1, and Calbindin-D9k in the small intestine. These results suggest that COS–SPs–Ca is a promising calcium supplement.

Eucommia ulmoides extract regulates oxidative stress to maintain calcium homeostasis and improve diabetic osteoporosis.

Diabetic osteoporosis (DOP) is a secondary disease that severely affects the health and quality of life of patients with diabetes mellitus. This study aimed to explore the bone protective effect of aqueous extract of Eucommia ulmoides (EUL) in DOP mice. DOP mice were established using a high-sugar, high-fat diet and streptozotocin (STZ) (35 mg/kg for three consecutive days), and the EUL aqueous extract (2.5 g/kg/day) was orally administered for 6 weeks. The serum levels of oxidative stress-related factors, calcium, and phosphorus were assessed using biochemical assays. The osteoprotective effect of EUL was assessed using micro-computer tomography, three-point bending assay, histological analysis, and immunoblotting. Quantitative real-time polymerase chain reaction and western blotting were performed to detect the expression levels of calcium transport channel factors in the kidney and small intestine tissues. Furthermore, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the femur, kidney, and small intestine tissues were detected using western blotting and quantitative real-time polymerase chain reaction. EUL aqueous extract reduced blood glucose levels, increased body weight, and relieved symptoms in DOP mice (p < .05). It also increased bone mineral density, improved the bone microstructure, decreased the number of femoral osteoclasts, and increased the expression of femoral Runx2 and Bmp2 in DOP mice (p < .01). After 6 weeks of EUL aqueous extract administration, serum levels of SOD, CTA, calcium, and phosphorus were upregulated, whereas MDA levels were decreased (p < .01). The aqueous EUL extract also upregulated the expression of TRPV5, PMCA-1b, and CaBP-9 k in the kidney and small intestine of DOP mice (p < .01). Furthermore, the expression of Nrf2 and HO-1 in the kidney, small intestine, and femur tissues was increased (p < .01). EUL aqueous extract reduced blood glucose levels in DOP mice and regulated oxidative stress through the Nrf2/HO-1 pathway, thereby maintaining calcium homeostasis and ultimately improving bone quality. Our study suggested that EUL aqueous extract may be effective in the treatment of DOP.

Impact of Lactobacillus acidophilus and Its Combination with Isoflavone Products on Calcium Status, Calcium Transporters, and Bone Metabolism Biomarkers in a Post-Menopausal Osteoporotic Rat Model.

Osteoporosis in menopausal women requires alternatives to current medications, considering their adverse effects. In this context, probiotics and isoflavone products are promising dietary interventions. The objective of our study was to examine the impacts of Lactobacillus acidophilus and its combination with daidzein and tempeh on calcium status, calcium transporters, and bone metabolism biomarkers in a post-menopausal osteoporotic rat model. A total of 48 female Wistar rats were exposed to a two-stage experiment involving calcium deficit induction and subsequent dietary interventions across six groups. Calcium levels, the gene expression of TRPV5 and TRPV6 calcium transporters, bone histopathology, serum bone metabolism markers, and blood biochemistry were evaluated. The results revealed that, while decreasing serum calcium levels, the groups that received the probiotic L. acidophilus and isoflavone combination exhibited increased bone metabolism biomarkers and decreased calcium transporter expressions, akin to the effects of bisphosphonate. Additionally, significant improvements in bone histopathology were observed in these groups. However, the group receiving probiotic L. acidophilus alone did not exhibit significant changes in bone resorption biomarkers, calcium transporter expression, or various blood parameters. Meanwhile, the combination of probiotic L. acidophilus with tempeh positively influenced hematological parameters and reduced cholesterol and triglyceride levels, but it led to elevated blood glucose levels. Correlation analyses highlighted associations between serum calcium levels, calcium transporter expression, and bone metabolism biomarkers. In conclusion, our findings suggest that the daily consumption of probiotic L. acidophilus in combination with isoflavone products may improve bone health in ovariectomized rats, warranting further research to elucidate potential interactions with other nutrients.

Curcumin ameliorates pyroptosis in diabetic seminal vesicles by upregulating TRPV6.

Diabetes damages the seminal vesicle tissues leading to a decrease in seminal fluid secretion, so investigations are ongoing to identify specific therapeutic approaches to address diabetes-induced damage to seminal vesicles. This study investigated the secretory dysfunction of seminal vesicles and how curcumin can ameliorate this dysfunction. First, 40 diabetic males (DM group) and 40 nondiabetic males (control group) underwent seminal vesicle ultrasound evaluation and ejaculate volume measurements. Then, the effects of curcumin on seminal vesicle function were investigated in a diabetic rat model. Fifty 8-week-old SPF-grade SD rats were categorized into five groups: control, DM (diabetes mellitus), low-dose CUR (curcumin 50mg/kg/d), medium-dose CUR (curcumin 100mg/kg/d), and high-dose CUR (curcumin 150mg/kg/d). After a month-long diet with varying curcumin doses, key parameters such as body weight, blood glucose levels, seminal vesicle volume, and seminal fluid secretion were measured. Transcriptome sequencingwas performed to assess differences in gene expression and structural changes in rat seminal vesicle tissues were examined by HE staining. Finally, human seminal vesicle cell lines were cultured and divided into five groups (HG-CON, HG-CUR-5µM, HG-CUR-10µM, HG-CUR-20µM, and HG-CUR-50µM) to measure the fructose levels in the seminal vesicle cell culture fluids and evaluate the expression of CASP1, GSDMD, and TRPV6. Post TRPV6 interference, variations in the gene expression of CASP1, GSDMD, and TRPV6 were monitored. Diabetic patients exhibited a notable reduction in seminal vesicle volume and ejaculate volume compared with the control group, with a direct correlation between the decrease in ejaculate and seminal vesicle volume. Animal studies demonstrated that curcumin supplementation significantly augmented seminal vesicle volume in diabetic rats and notably improved their seminal vesicle secretory dysfunction, particularly in the high-dose curcumin group. Transcriptome sequencing and experimental verification pinpointed the differential expression of TPRV6 and pyroptosis-associated genes (CASP1, GSDMD), with reduced TRPV6 expression but increased markers of pyroptosis (CASP1 and GSDMD) in diabetic rats. Curcumin treatment reversed these effects with an increase in TRPV6 and a decrease in GSDMD and CASP1. Cell transfection experiments indicated that TRPV6 downregulation increased GSDMD and CASP1 gene expression. Curcumin effectively activates TRPV6, thereby diminishing pyroptosis in the seminal vesicle tissues of diabetic rats. This activation not only leads to an increase in the seminal vesicle volume but also significantly ameliorates the seminal vesicle secretory dysfunction in diabetic rats.

Characterization and Mechanism of a Novel Rice Protein Peptide (AHVGMSGEEPE) Calcium Chelate in Enhancing Calcium Absorption in Caco-2 Cells.

Rice protein peptides (RPP) are a potentially valuable source of high-quality calcium chelating properties. However, there is a lack of information regarding the calcium-absorption-promoting effect of RPP and its underlying mechanism. The present study adopted molecular docking methodologies to analyze the 10 most potent peptide segments from RPP. Results revealed that the peptide AHVGMSGEEPE (AHV) displayed optimal calcium binding properties (calcium-chelating capacity 55.69 ± 0.66 mg/g). Quantum chemistry analysis revealed that the AHV peptide effectively binds and forms stable complexes with calcium via the carbonyl oxygen atoms in valine at position 3 and the carbonyl of the C-terminal carboxyl group of glutamate at position 11. The spectral analysis results indicated that AHV may bind to calcium through carboxyl oxygen atoms, resulting in a transition from a smooth surface block-like structure to a dense granular structure. Furthermore, this study demonstrated that the 4 mmol/L AHV-Ca chelate (61.75 ± 13.23 μg/well) significantly increases calcium absorption compared to 1 mM CaCl2 (28.57 ± 8.59 μg/well) in the Caco-2 cell monolayer. In terms of mechanisms, the novel peptide-calcium chelate AHV-Ca derived from RPP exerts a cell-level effect by upregulating the expression of TRPV6 calcium-ion-channel-related genes and proteins (TRPV6 and Calbindin-D9k). This study provides a theoretical basis for developing functional foods with the AHV peptide as ingredients to improve calcium absorption.

Effect of Tempeh and Daidzein on Calcium Status, Calcium Transporters, and Bone Metabolism Biomarkers in Ovariectomized Rats.

Menopause marks a critical life stage characterized by hormonal changes that significantly impact bone health, leading to a heightened susceptibility to bone fractures. This research seeks to elucidate the impact of daidzein and tempeh on calcium status, calcium transporters, and bone metabolism in an ovariectomized rat model. Forty female Wistar rats, aged 3 months, participated in a two-phase experiment. The initial phase involved inducing a calcium deficit, while the second phase comprised dietary interventions across five groups: Sham (S) and Ovariectomy (O) with a standard diet, O with bisphosphonate (OB), O with pure daidzein (OD), and O with tempeh (OT). Multiple parameters, encompassing calcium levels, calcium transporters, bone histopathology, and serum bone metabolism markers, were evaluated. The findings revealed that the OT group showcased heightened levels of bone turnover markers, such as pyridinoline, C-telopeptide of type I collagen, bone alkaline phosphatase, and procollagen type I N-terminal propeptide, in contrast to S and O groups, with statistical significance (p < 0.05). Histopathologically, both the OD and OT groups exhibited effects akin to the OB group, indicating a decrease in the surface area occupied by adipocytes in the femoral bone structure, although statistically non-equivalent, supporting the directionally similar trends. Although TRPV5 and TRPV6 mRNA expression levels in the jejunum and duodenum did not display statistically significant differences (p > 0.05), the OD and OT groups exhibited increased expression compared to the O group. We hypothesized that obtained results may be related to the effect of isoflavones on estrogen pathways because of their structurally similar to endogenous estrogen and weak estrogenic properties. In conclusion, the daily consumption of pure daidzein and tempeh could potentially improve and reinstate calcium status, calcium transport, and bone metabolism in ovariectomized rats. Additionally, isoflavone products demonstrate effects similar to bisphosphonate drugs on these parameters in ovariectomized rats.

Relationship of the bone phenotype of the Klotho mutant mouse model of accelerated aging to changes in skeletal architecture that occur with chronological aging.

Due to the relatively long life span of rodent models, in order to expediate the identification of novel therapeutics of age related diseases, mouse models of accelerated aging have been developed. In this study we examined skeletal changes in the male and female Klotho mutant (kl/kl) mice and in male and female chronically aged mice to determine whether the accelerated aging bone phenotype of the kl/kl mouse reflects changes in skeletal architecture that occur with chronological aging. 2, 6 and 20-23 month old C57BL/6 mice were obtained from the National Institute of Aging aged rodent colony and wildtype and kl/kl mice were generated as previously described by M. Kuro-o. Microcomputed tomography analysis was performed ex vivo to examine trabecular and cortical parameters from the proximal metaphyseal and mid-diaphyseal areas, respectively. Serum calcium and phosphate were analyzed using a colorimetric assay. The expression of duodenal Trpv6, which codes for TRPV6, a vitamin D regulated epithelial calcium channel whose expression reflects intestinal calcium absorptive efficiency, was analyzed by quantitative real-time PCR. Trabecular bone volume (BV/TV) and trabecular number decreased continuously with age in males and females. In contrast to aging mice, an increase in trabecular bone volume and trabecular number was observed in both male and female kl/kl mice. Cortical thickness decreased with advancing age and also decreased in male and female kl/kl mice. Serum calcium and phosphate levels were significantly increased in kl/kl mice but did not change with age. Aging resulted in a decline in Trpv6 expression. In the kl/kl mice duodenal Trpv6 was significantly increased. Our findings reflect differences in bone architecture as well as differences in calcium and phosphate homeostasis and expression of Trpv6 between the kl/kl mutant mouse model of accelerated aging and chronological aging. Although the Klotho deficient mouse has provided a new understanding of the regulation of mineral homeostasis and bone metabolism, our findings suggest that changes in bone architecture in the kl/kl mouse reflect in part systemic disturbances that differ from pathophysiological changes that occur with age including dysregulation of calcium homeostasis that contributes to age related bone loss.

Effect of Vitamin D overdose on VDR, TRPV6 and CYP3A11 genes expression, biochemical tests and histopathological legions in Albino mice

Effect of Vitamin D overdose on VDR, TRPV6 and CYP3A11 genes expression, biochemical tests and histopathological legions in Albino mice

Effects of enzyme supplementation on growth performance, digestibility of phosphorus, femur parameters and fecal microbiota in growing pigs fed different types of diets.

A 42-days study was conducted to evaluate the effects of different dietary types (corn-or wheat-soybean meal-based diet) and phytase (Phy) or a multi-carbohydrase and phytase complex (MCPC) supplementation on growth performance, digestibility of phosphorus (P), intestinal transporter gene expression, plasma indexes, bone parameters, and fecal microbiota in growing pigs. Seventy-two barrows (average initial body weight of 24.70 ± 0.09 kg) with a 2 × 3 factorial arrangement of treatments and main effects of diet type (corn-or wheat-soybean meal-based-diets) and enzyme supplementation (without, with Phy or with MCPC). Each group was designed with 6 replicate pens. The MCPC increased (p < 0.05) average daily gain (ADG) and final body weight (BW). A significant interaction (p = 0.01) was observed between diet type and enzyme supplementation on apparent total tract digestibility (ATTD) of P. The ATTD of P was higher (p < 0.05) in wheat soybean meal-based diets compared to corn-soybean meal-based diets. Compared with the corn-soybean meal-based diet, the relative expression of SLC34A2 and VDR genes in the ileum and SLC34A3 in jejunum of growing pigs fed the wheat-soybean meal based diet was lower (p < 0.05). The MCPC significantly reduced (p < 0.05) the relative expression of TRPV5 and CALB1 genes in the ileum and increased the expression of CALB1 in the duodenum compared to control diet. The phytase increased (p < 0.05) the relative expression of SLC34A1 gene in the duodenum in comparison to control diet and MCPC-supplemented diet. The Ca and P contents in plasma from pigs fed corn-soybean meal-based diet were higher (p < 0.05) than those from pigs fed wheat-soybean meal-based diet, and the parathyroid hormone (PTH) and calcitonin (CT) concentrations were lower (p < 0.05) than those fed wheat-soybean meal-based diet. The content of Ca and P in the femur and the bone strength of pigs in the corn-soybean meal group were significantly higher (p < 0.05) than those in the wheat-soybean meal groups. The phytase increased (p < 0.05) the Ca and P content and bone strength of the femur. Additionally, diet type and both enzymes significantly improved fecal microbial diversity and composition. Taken together, diet type and exogenous enzymes supplementation could differently influence the growth performance, utilization of phosphorus, intestinal transporter gene expression, bone mineralization and microbial diversity and composition in growing pigs.

TRPV6 Channel Is Involved in Pancreatic Ductal Adenocarcinoma Aggressiveness and Resistance to Chemotherapeutics.

Pancreatic ductal adenocarcinoma (PDAC) stands as a highly aggressive and lethal cancer, characterized by a grim prognosis and scarce treatment alternatives. Within this context, TRPV6, a calcium-permeable channel, emerges as a noteworthy candidate due to its overexpression in various cancers, capable of influencing the cell behavior in different cancer entities. Nonetheless, the exact expression pattern and functional significance of TRPV6 in the context of PDAC remains enigmatic. This study scrutinizes the expression of TRPV6 in tissue specimens obtained from 46 PDAC patients across distinct stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. Subsequently, we analyzed its impact on multiple facets, encompassing Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth, both in vitro and in vivo. Notably, the data indicate a direct correlation between TRPV6 expression levels, tumor stage, and grade, establishing a link between TRPV6 and PDAC proliferation in tissue samples. Decreasing TRPV6 expression via knockdown hampered Ca2+ influx, resulting in diminished proliferation and viability in both cell lines, and cell cycle progression in Panc-1. The knockdown simultaneously led to an increase in apoptotic rates and increased the susceptibility of cells to 5-FU and gemcitabine treatments. Moreover, it accelerated migration and promoted collective movement among Panc-1 cells. Conversely, TRPV6 overexpression yielded opposing outcomes in terms of proliferation in Panc-1 and Capan-1, and the migration of Panc-1 cells. Intriguingly, both TRPV6 knockdown and overexpression diminished the process of tumor formation in vivo. This intricate interplay suggests that PDAC aggressiveness relies on a fine-tuned TRPV6 expression, raising its profile as a putative therapeutic target.

Stanniocalcin 1a regulates organismal calcium balance and survival by suppressing Trpv6 expression and inhibiting IGF signaling in zebrafish.

Stanniocalcin 1 (Stc1) is well known for its role in regulating calcium uptake in fish by acting on ionocytes or NaR cells. A hallmark of NaR cells is the expression of Trpv6, a constitutively open calcium channel. Recent studies in zebrafish suggest that genetical deletion of Stc1a and Trpv6 individually both increases IGF signaling and NaR cell proliferation. While trpv6-/- fish suffered from calcium deficiency and died prematurely, stc1a-/- fish had elevated body calcium levels but also died prematurely. The relationship between Stc1a, Trpv6, and IGF signaling in regulating calcium homeostasis and organismal survival is unclear. Here we report that loss of Stc1a increases Trpv6 expression in NaR cells in an IGF signaling-dependent manner. Treatment with CdCl2, a Trpv6 inhibitor, reduced NaR cell number in stc1a -/- fish to the sibling levels. Genetic and biochemical analysis results suggest that Stc1a and Trpv6 regulate NaR cell proliferation via the same IGF pathway. Alizarin red staining detected abnormal calcium deposits in the yolk sac region and kidney stone-like structures in stc1a -/- fish. Double knockout or pharmacological inhibition of Trpv6 alleviated these phenotypes, suggesting that Stc1a inhibit epithelial Ca2+ uptake by regulating Trpv6 expression and activity. stc1a-/- mutant fish developed cardiac edema, body swelling, and died prematurely. Treatment of stc1a-/- fish with CdCl2 or double knockout of Trpv6 alleviated these phenotypes. These results provide evidence that Stc1a regulates calcium homeostasis and organismal survival by suppressing Trpv6 expression and inhibiting IGF signaling in ionocytes.

Vitamin D3 mediated peptides-calcium chelate self-assembly: Fabrication, stability and improvement on cellular calcium transport

A self-assembled peptides-calcium-Vitamin D3 ternary delivery system (CSPH-Ca-VD3) was prepared to investigate the promotion of cellular calcium transport. The constructed CSPH-Ca-VD3 nanocomplex exhibited a spherical structure with a size of 135.2 ± 10.2 nm. Based on the thermodynamic calculation of fluorescent spectra, hydrophobic interaction was shown as the major driving force for this nanocomplex structure. CSPH-Ca-VD3 nanocomplex possessed excellent stability during simulated gastrointestinal digestion, contributing to the prevention of acid degradation of VD3 and the enhancement of calcium solubility. Furthermore, the calcium transport efficiency in the form of CSPH-Ca-VD3 (4 mg/mL) across a Caco-2 cells monolayer was significantly increased 2.3-fold compared to that of free Ca2+, mainly attributed to the upregulation in the presence of CSPH-Ca-VD3 of TRPV6, calbindin D9k and PMCA1b expression in Caco-2 cells. The present study provided a basis for developing a novel delivery system of peptides-calcium chelate with the dual effects of VD3 protection and calcium uptake promotion.

Prepubertal to adulthood exposure to low doses of glyphosate-based herbicide increases the expression of the Havcr1 (Kim1) biomarker and causes mild kidney alterations

Glyphosate is a nonselective and postemergent herbicide used to combat weeds in several crops, which raises concerns about risks to human health since residues are detected in urine, human milk, surface water and several types of food. Feces and urine are the major routes of elimination of glyphosate, making the kidney a sensitive target for the development of toxicity. In fact, farmers are at high risk of developing chronic kidney disease. In this sense, this study aims to investigate kidney function by measuring the serum levels of urea and creatinine, examining the histological morphology, and analyzing the mRNA expression of genes related to tubular transport of ions, urea and urates and the biomarker of kidney disease Kim1, and the levels of lead in the kidney in male Wistar rats orally exposed to low levels of glyphosate-based herbicide (GBH: 0, 0.5 or 5 mg/kg) from weaning to adult life by gavage. GBH0.5 showed reduced serum urea concentration, presence of tubulointerstitial swelling and mononuclear cell infiltration into the interstitium, increased gene expression of Kim1 and reduced gene expression of Slc14a1. GBH5 showed reduced serum urea and increased serum creatinine concentrations, tubulointerstitial swelling, interstitial fibrosis, and reduced expression of Trpm6 and Trpv5. Exposure to GBH did not affect the levels of Pb in the kidneys of animals. In conclusion, glyphosate at low doses may cause mild kidney damage. It is necessary to evaluate whether the long-term effects of this constant injury may contribute to the development of chronic kidney disease of uncertain etiology.

A novel frameshift mutation in TRPV6 is associated with hereditary pancreatitis.

Introduction: Hereditary pancreatitis (HP) is a rare debilitating disease with incompletely understood etio-pathophysiology. The reduced penetrance of genes such as PRSS1 associated with hereditary pancreatitis indicates a role for novel inherited factors. Methods: We performed whole-exome sequencing of three affected members of an Indian family (Father, Son, and Daughter) with chronic pancreatitis and compared variants with those seen in the unaffected mother. Results: We identified a novel frameshift mutation in exon 11 of TRPV6 (c.1474_1475delGT; p.V492Tfs*136), a calcium channel, in the patients. Functional characterization of this mutant TRPV6 following heterologous expression revealed that it was defective in calcium uptake. Induction of pancreatitis in mice induced Trpv6 expression, indicating that higher expression levels of the mutant protein and consequent dysregulation of calcium levels in patients with chronic pancreatitis could aggravate the disease. Discussion: We report a novel frameshift mutation in TRPV6 in an Indian family with HP that renders the mutant protein inactive. Our results emphasize the need to expand the list of genes used currently for evaluating patients with hereditary pancreatitis.

Disrupted placental vitamin D metabolism and calcium signaling in gestational diabetes and pre-eclampsia patients.

Gestational diabetes (GDM) and pre-eclampsia (PE) represents the unrecognized risk factors for reduced bone content in neonates. The present study is planned to explore the components of vitamin D metabolism and calcium transport in placenta of GDM and PE cases and its effect on the neonatal bone mass determination using bone densitometry system. We have collected serum and placenta tissues from GDM (n = 20), PE (n = 20), and healthy pregnancies (n = 20). In the present study, we found mRNA expression of oxidative stress markers, vitamin D metabolic components and calcium channels, calcium channel binding proteins, plasma membrane calcium ATPase, ATP synthase and Ca2+ release genes; Ryanodine receptors genes were assessed by quantitative real-time PCR (qRT-PCR) in placental tissue of GDM, PE, and healthy pregnancies. We observed high level of oxidative stress in both GDM and PE placenta compared to normal pregnancies. CYP2R1 and VDR mRNA expression was significantly downregulated and upregulation of CYP27B1 and CYP24A1 in GDM and PE compared with healthy cases. Similarly, calcium transporters were downregulated in GDM and PE placental tissues. In addition, CYP24A1, VDR, CaBP28K, TRPV5 and PMCA3 mRNA expression were correlated with BMC of neonates. Oxidative stress is probably relevant to disrupted vitamin D homeostasis and calcium transport in the placenta of GDM and PE cases. The altered regulatory mechanism of CYP24A1 and VDR could indicates more pronounced serum 25(OH)D reduction. Additionally, reduced BMC in the neonates of these cases might be as consequences of modified CYP24A1, VDR, CaBP28K, TRPV5 and PMCA3 mRNA expression.

PSUN112 Expression of Vitamin D Receptor Pathway Genes Across Tissues of Individuals with Obesity

Abstract Introduction Vitamin D deficiency is a substantial comorbidity in 50% of pediatric patients and is linked with poorer health outcomes in children. Vitamin D levels are inversely related to BMI. Therefore, the prevalence of children with low vitamin D levels is increasing as the prevalence of pediatric obesity increases. Pediatric patients with obesity and vitamin D deficiency also have a uniquely increased risk of metabolic syndrome, as compared to their lean peers. However, measured levels of vitamin D correlate with other physiological markers of vitamin D effects in lean individuals but not obese individuals. Since vitamin D levels measured as 25-hydroxyvitmain D (serum 25(OH)D) reflect a storage form of vitamin D, their levels may not be a true reflection of vitamin D action in the body in an obese population. The aim of this study was to provide foundational knowledge to understand if expression of vitamin D receptor (VDR)-target genes in different metabolic pathways correlate with each other in diverse tissues, and thus may be used as a reference standard for vitamin D action in the body. Methods We performed a secondary analysis of samples obtained from 89 obese adolescents aged 12 to 18 years old that were consented under a past IRB-approved protocol. The samples were collected at the time of bariatric surgery between 2004 and 2019. Subject data included age, gender, race/ethnicity, and BMI. Samples collected included blood, intestinal, and subcutaneous adipose tissue. The tissues were analyzed via Real Time-PCR to obtain quantitative levels of VDR-target gene expression, which included TLR4, THBD, and VDR in subcutaneous adipose tissue; and TRPV6, S100G, and VDR in intestinal tissue. Gene expression levels were normalized to the average of two housekeeping genes, GAPDH and RPLPO. Blood samples were analyzed for vitamin D levels. Results VDR-target gene expression of THBD, VDR, and TLR4 in subcutaneous adipose tissue was significantly correlated (p &amp;lt;0.05). In intestinal tissue, we also saw significant correlation between TRPV6, S100G, and VDR (p &amp;lt;0.05). THBD, TLR4, TRPV6, S100G, and VDR gene expression levels from the respective tissues did not correlate with circulating serum 25(OH)D levels (p &amp;gt;0.05). Conclusion These important preliminary findings show VDR-target genes have correlated expression patterns within different tissues – one which stores vitamin D (adipose) and one which doesn't (intestinal). Interestingly, the VDR-target gene expression levels correlated with each other despite the genes being involved in different metabolic pathways in diverse tissues. These findings also show that VDR-target gene expression does not correlate with circulating serum 25(OH)D levels. This discrepancy supports that 25(OH)D levels do not indicate levels of vitamin D action and may not be an appropriate indicator of vitamin D deficiency in the obese population. Thus, VDR-target gene expression levels may provide a better reference standard for vitamin D action in the body. This study may provide the first step in determining a new and more accurate biomarker for vitamin D deficiency and treatment in obesity. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Aerobic exercise combined with glucosamine hydrochloride capsules inhibited the apoptosis of chondrocytes in rabbit knee osteoarthritis by affecting TRPV5 expression

ObjectiveThis study aimed to investigate the effect of aerobic exercise combined with glucosamine (OTL) on the apoptosis of chondrocytes of rabbit knee osteoarthritis (KOA) by affecting the expression of TRPV5. MethodsAfter the KOA white rabbit model was established, aerobic training and OTL treatment were performed, then the model joints were evaluated by Mankin, HE staining was used to observe the pathological changes of articular cartilage, TUNEL and immunohistochemistry were used to detect chondrocyte apoptosis. Knee chondrocytes were isolated and identified by Alcian Blue and type II collagen fiber staining. The cells were treated with iodoacetic acid (MIA) to simulate osteoarthritis in vitro, and then the effect of TRPV5 on apoptosis was detected by flow cytometry, in addition, apoptosis-related proteins and TRPV5 were detected by western blotting and qRT-PCR. ResultsBoth aerobic exercise and OTL treatment could significantly reduce the Mankin score of KOA model, and could effectively inhibit chondrocyte apoptosis in the KOA model, and inhibit the expression of caspase 3 and caspase 9 in the KOA model. TRPV5 expression was significantly increased in the model, while both aerobic exercise and OTL could reverse its expression. The low-expression of TRPV5 significantly reversed the role of MIA in promoting apoptosis and apoptosis-related proteins of knee chondrocytes, while overexpressing TRPV5 promoted MIA-induced apoptosis and apoptosis-related proteins. ConclusionAerobic exercise combined with glucosamine hydrochloride capsules inhibited the apoptosis of chondrocytes in rabbit KOA by affecting the expression of TRPV5.