Calcium Reabsorption Research Articles

SINGLE-NUCLEOTIDE POLYMORPHISMS OF CALCIUM-SENSING RECEPTOR ENCODING GENE ASSOCIATED WITH CALCIUM KIDNEY STONE DISEASE IN BABYLON PROVINCE

Objective: The calcium-sensing receptor (CASR) is a G-protein-coupled receptor that is mainly expressed in the parathyroid and the kidneys where it regulates parathyroid hormone secretion and renal tubular calcium reabsorption. Inactivating and activating CASR gene due to mutations severally caused hypercalcemia or hypocalcemia disorders. The aim of the study was to investigate the risk factor of CASR rs1801725 (Ala986Ser) patients with renal disease.Method: The blood samples were collected from 100 patients and divided into two groups, each one containing 50 samples; chronic kidney disease and end-stage renal disease, who admitted Merjan Teaching Hospital in Babylon Province, Iraq, from February to July 2016. In addition, healthy persons as a control group (50 samples). Genotyping of CASR single-nucleotide polymorphisms (SNP) was performed using a polymerase chain reaction technique, followed by single-strand conformation polymorphism. Accordingly, these DNA polymorphisms were confirmed using DNA sequencing.Results: The conformational haplotypes of CASR, exon7 NCBI Primer3plus reference were obtained in three patterns, including two, three, and four bands, due to the presence SNPs within the studied region. These SNPs leads to change three amino acid residues of CASR, including amino acid substitutions were Ala 128→ Ser 128, Leu 155→Tye 155, and Leu 156→ Ser 156 that may affect or modified the tertiary structure of the receptor, subsequently the function like the affinity to calcium ion may be effected.Conclusion: These results suggest that the variants of CASR SNP, namely, rs1801725 might be involved in susceptibility to kidney stone disease.

Rickets/Osteomalacia. The function and mechanism of vitamin D action.

The antirickets factor vitamin D exhibits its action through activation of vitamin D receptor(VDR). The active form of vitamin D, 1α,25-dihydroxyvitamin D3[1,25(OH)2D3], is a potent VDR ligand, and contributes to the maintenance of calcium homeostasis by enhancing intestinal calcium absorption, renal calcium reabsorption and bone resorption. 1,25(OH)2D3 also regulates bone formation, phosphorus metabolism and vitamin D metabolism. Experimental and epidemiological evidence has shown vitamin D actions on cellular proliferation and differentiation, immunity and inflammation, and cardiovascular function. Vitamin D derivatives and VDR ligands may be useful for the treatment of osteoporosis, malignancy, autoimmune and inflammatory disease and cardiovascular disease as well as rickets and osteomalacia.

Physiology of the Calcium-Parathyroid Hormone-Vitamin D Axis.

Classic endocrine feedback loops ensure the regulation of blood calcium. Calcium in the extracellular fluid (ECF) binds and activates the calcium sensing receptor (CaSR) on the parathyroid cells, leading to an increase in intracellular calcium. This in turn leads to a reduced parathyroid hormone (PTH) release. Hypocalcemia leads to the opposite sequence of events, namely, lowered intracellular calcium and increased PTH production and secretion. PTH rapidly increases renal calcium reabsorption and, over hours to days, enhances osteoclastic bone resorption and liberates both calcium and phosphate from the skeleton. PTH also increases fibroblast growth factor 23 (FGF23) release from mature osteoblasts and osteocytes. PTH stimulates the renal conversion of 25-hydroxyvitamin D (25[OH]D) to 1,25(OH)2D, likely over several hours, which in turn will augment intestinal calcium absorption. Prolonged hypocalcemia and exposure to elevated PTH may also result in 1,25(OH)2D-mediated calcium and phosphorus release from bone. These effects restore the ECF calcium to normal and inhibit further production of PTH and 1,25(OH)2D. Additionally, FGF23 can be released from bone by 1,25(OH)2D and can in turn reduce 1,25(OH)2D concentrations. FGF23 has also been reported to decrease PTH production. When ECF calcium is in the hypercalcemic range, PTH secretion is reduced and renal 1,25(OH)2D production is decreased. In addition, the elevated calcium per se stimulates the renal CaSR, thus inducing calciuria. Therefore, suppression of PTH release and 1,25(OH)2D synthesis and stimulation of the renal CaSR lead to reduced renal calcium reabsorption, decreased skeletal calcium release, and decreased intestinal calcium absorption, resulting in the normalization of the elevated ECF calcium.

Abaloparatide, a novel PTH receptor agonist, increased bone mass and strength in ovariectomized cynomolgus monkeys by increasing bone formation without increasing bone resorption

SummaryAbaloparatide, a novel PTH1 receptor agonist, increased bone formation in osteopenic ovariectomized cynomolgus monkeys while increasing cortical and trabecular bone mass. Abaloparatide increased bone strength and maintained or enhanced bone mass-strength relationships, indicating preserved or improved bone quality.IntroductionAbaloparatide is a selective PTH1R activator that is approved for the treatment of postmenopausal osteoporosis. The effects of 16 months of abaloparatide administration on bone formation, resorption, density, and strength were assessed in adult ovariectomized (OVX) cynomolgus monkeys (cynos).MethodsSixty-five 9–18-year-old female cynos underwent OVX surgery, and 15 similar cynos underwent sham surgery. After a 9-month period without treatments, OVX cynos were allocated to four groups that received 16 months of daily s.c. injections with either vehicle (n = 17) or abaloparatide (0.2, 1, or 5 μg/kg/day; n = 16/dose level), while Sham controls received s.c. vehicle (n = 15). Bone densitometry (DXA, pQCT, micro-CT), qualitative bone histology, serum calcium, bone turnover markers, bone histomorphometry, and bone strength were among the key measures assessed.ResultsAt the end of the 9-month post-surgical bone depletion period, just prior to the treatment phase, the OVX groups exhibited increased bone turnover markers and decreased bone mass compared with sham controls. Abaloparatide administration to OVX cynos led to increased bone formation parameters, including serum P1NP and endocortical bone formation rate. Abaloparatide administration did not influence serum calcium levels, bone resorption markers, cortical porosity, or eroded surfaces. Abaloparatide increased bone mass at the whole body, lumbar spine, tibial diaphysis, femoral neck, and femoral trochanter. Abaloparatide administration was associated with greater lumbar vertebral strength, and had no adverse effects on bone mass-strength relationships for the vertebrae, femoral neck, femoral diaphysis, or humeral cortical beams.ConclusionsAbaloparatide administration was associated with increases in bone formation, bone mass and bone strength, and with maintenance of bone quality in OVX cynos, without increases in serum calcium or bone resorption parameters.

Comparative transcriptomic analysis of high and low egg-producing duck ovaries

The egg-laying rate is an important indicator of egg production of laying ducks. Egg production directly impacts the economic benefits of the duck industry. In order to obtain better insight into the molecular mechanisms associated with the process of egg production, comparative transcriptomic analysis of the ovaries of Jinding ducks with high and low egg production was performed using the Illumina HiSeq 2500 system. A total of 843 differentially expressed genes (DEGs) was identified, 367 that were down-regulated and 476 that were up-regulated in high egg production (HEP) ovaries, as compared with low egg production (LEP) ovaries. Some genes, such as MC5R, APOD, ORAI1, and DYRK4, were more active in HEP ovaries, indicating that these genes may play important roles in regulation of egg production. Among these 843 DEGs, 685 were assigned to gene ontology (GO) categories. Of these, 25 genes were related to reproduction, and 30 were related to the reproductive process, including some associated with ovarian follicle development, circadian regulation of gene expression, circadian rhythm, and estrogen receptor binding. Furthermore, some important functional pathways were revealed, such as the steroid biosynthesis pathway, the endocrine and other factor-regulated calcium reabsorption pathways, circadian rhythm, the neuroactive ligand-receptor interaction pathway, fatty acid biosynthesis, and the calcium-signaling pathway, which appear to be much more active in the HEP group, as compared to those of the LEP group. The results of this study provide very useful information that may contribute to future functional studies of genes involved in bird reproduction.

Familial Hypocalciuric Hypercalcemia as an Atypical Form of Primary Hyperparathyroidism.

Familial hypocalciuric hypercalcemia (FHH) causes lifelong hypercalcemia with features that overlap with typical primary hyperparathyroidism (PHPT). The incompleteness of this overlap has led to divergent nomenclatures for FHH. I compare two nomenclatures. One sets FHH as an entity distinct from PHPT. The other groups FHH with PHPT but conditions FHH as atypical PHPT. I analyzed selected articles about calcium-sensing receptors, FHH, PHPT, CASR, GNA11, and AP2S1. FHH usually results from a heterozygous germline inactivating mutation of the CASR, and less frequently from mutation of GNA11 or AP2S1. The CASR encodes the calcium-sensing receptors. These are highly expressed on parathyroid cells, where they sense serum calcium concentration and regulate suppression of PTH secretion by serum calcium. Their mutated expression in the kidney in FHH causes increased renal tubular reabsorption of calcium (hypocalciuria). Many FHH features are shared with PHPT and thus support FHH as a form of PHPT. These include a driver mutation expressed mainly in the parathyroid cells. The mutation causes a parathyroid cell insensitivity to extracellular calcium in vivo and in vitro, a right-shift of the set point for suppression of PTH secretion by calcium. Serum PTH is normal or mildly elevated; ie, it is not appropriately suppressed by hypercalcemia. Total parathyroidectomy causes hypoparathyroidism and durable remission of hypercalcemia. Some other features are not shared with PHPT and could support FHH as a distinct entity. These include onset of hypercalcemia in the first week of life, frequent persistence of hypercalcemia after subtotal parathyroidectomy, and hypocalciuria. The features supporting FHH as a form of PHPT are stronger than those favoring FHH as a distinct entity. Classifying FHH as an atypical form of PHPT represents compact nomenclature and supports current concepts of pathophysiology of FHH and PHPT. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Calciuria as a metabolic marker for various conditions and diseases

The article analyzes the literature on the features of human calcium homeostasis. The authors describe the etiopathogenetic role of calcitropic hormones, the plasma and urine acid-base status, various ions, lifestyle and nutrition and other factors contributing to hypercalciuria due to increased intestinal absorption, bone resorption, impairment of tubular calcium reabsorption, etc. They discuss the role of calciuria as a factor in forming urinary calculi and present their own observations.

A Network Pharmacology Approach to Determine the Active Components and Potential Targets of Curculigo Orchioides in the Treatment of Osteoporosis.

BackgroundOsteoporosis is a complex bone disorder with a genetic predisposition, and is a cause of health problems worldwide. In China, Curculigo orchioides (CO) has been widely used as a herbal medicine in the prevention and treatment of osteoporosis. However, research on the mechanism of action of CO is still lacking. The aim of this study was to identify the absorbable components, potential targets, and associated treatment pathways of CO using a network pharmacology approach.Material/MethodsWe explored the chemical components of CO and used the five main principles of drug absorption to identify absorbable components. Targets for the therapeutic actions of CO were obtained from the PharmMapper server database. Pathway enrichment analysis was performed using the Comparative Toxicogenomics Database (CTD). Cytoscape was used to visualize the multiple components-multiple target-multiple pathways-multiple disease network for CO.ResultsWe identified 77 chemical components of CO, of which 32 components could be absorbed in the blood. These potential active components of CO regulated 83 targets and affected 58 pathways. Data analysis showed that the genes for estrogen receptor alpha (ESR1) and beta (ESR2), and the gene for 11 beta-hydroxysteroid dehydrogenase type 1, or cortisone reductase (HSD11B1) were the main targets of CO. Endocrine regulatory factors and factors regulating calcium reabsorption, steroid hormone biosynthesis, and metabolic pathways were related to these main targets and to ten corresponding compounds.ConclusionsThe network pharmacology approach used in our study has attempted to explain the mechanisms for the effects of CO in the prevention and treatment of osteoporosis, and provides an alternative approach to the investigation of the effects of this complex compound.

Effects of phospho- and calciotropic hormones on electrolyte transport in the proximal tubule.

Calcium and phosphate are critical for a myriad of physiological and cellular processes within the organism. Consequently, plasma levels of calcium and phosphate are tightly regulated. This occurs through the combined effects of the phospho- and calciotropic hormones, parathyroid hormone (PTH), active vitamin D 3, and fibroblast growth factor 23 (FGF23). The organs central to this are the kidneys, intestine, and bone. In the kidney, the proximal tubule reabsorbs the majority of filtered calcium and phosphate, which amounts to more than 60% and 90%, respectively. The basic molecular mechanisms responsible for phosphate reclamation are well described, and emerging work is delineating the molecular identity of the paracellular shunt wherein calcium permeates the proximal tubular epithelium. Significant experimental work has delineated the molecular effects of PTH and FGF23 on these processes as well as their regulation of active vitamin D 3 synthesis in this nephron segment. The integrative effects of both phospho- and calciotropic hormones on proximal tubular solute transport and subsequently whole body calcium-phosphate balance thus have been further complicated. Here, we first review the molecular mechanisms of calcium and phosphate reabsorption from the proximal tubule and how they are influenced by the phospho- and calciotropic hormones acting on this segment and then consider the implications on both renal calcium and phosphate handling as well as whole body mineral balance.

Pleiotropic Actions of FGF23.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone, mainly produced by osteoblasts and osteocytes in response to increased extracellular phosphate and circulating vitamin D hormone. Endocrine FGF23 signaling requires co-expression of the ubiquitously expressed FGF receptor 1 (FGFR1) and the co-receptor α-Klotho (Klotho). In proximal renal tubules, FGF23 suppresses the membrane expression of the sodium–phosphate cotransporters Npt2a and Npt2c which mediate urinary reabsorption of filtered phosphate. In addition, FGF23 suppresses proximal tubular expression of 1α-hydroxylase, the key enzyme responsible for vitamin D hormone production. In distal renal tubules, FGF23 signaling activates with-no-lysine kinase 4, leading to increased renal tubular reabsorption of calcium and sodium. Therefore, FGF23 is not only a phosphaturic but also a calcium- and sodium-conserving hormone, a finding that may have important implications for the pathophysiology of chronic kidney disease. Besides these endocrine, Klotho-dependent functions of FGF23, FGF23 is also an auto-/paracrine suppressor of tissue-nonspecific alkaline phosphatase transcription via Klotho-independent FGFR3 signaling, leading to local inhibition of mineralization through accumulation of pyrophosphate. In addition, FGF23 may target the heart via an FGFR4-mediated Klotho-independent signaling cascade. Taken together, there is emerging evidence that FGF23 is a pleiotropic hormone, linking bone with several other organ systems.

Prepartum daylight exposure increases serum calcium concentrations in dairy cows at the onset of lactation.

In dairy cows, hypocalcemia is caused by the sudden calcium demand by the mammary gland at the onset of lactation. Calcitriol (1,25-dihydroxy vitamin D; 1,25-VitD) increases the intestinal calcium absorption and the renal calcium reabsorption. Daylight contributes to the formation of 1,25-VitD, as it transforms 7-dehydrocholesterol into cholecalciferol, a 1,25-VitD precursor. Calving pens are usually set in quiet places where cows can stay calm and relaxed before parturition. However, those pens often have poor lighting conditions and therefore cows may become vitamin D deficient because of inadequate daylight exposure. Therefore, we have tested the hypothesis that direct daylight exposure supports the synthesis of 1,25-VitD and consequently attenuates the decline of calcium concentrations at parturition. Twenty Holstein dairy cows were randomly assigned to 2 experimental groups (daylight group, DL; and control group). Beginning on d -10 before expected parturition, both groups were placed in a standard indoor calving pen from 1700 h to 800 h. From 800 h to 1700 h cows from the DL group were moved into a contiguous open pen with direct access to daylight whereas controls remained at the standard indoor calving pen. After parturition both groups were permanently placed in an indoor calving pen until the end of the experimental period (d 30 postpartum). Blood samples were collected daily from d -10 prior to expected parturition through d 7 postpartum with an additional sample on d 30 postpartum. Milk yield was recorded at each milking during the whole experimental period. In the DL group, 25-hydroxyvitamin D (25-VitD), 1,25-VitD and total calcium concentrations around parturition were higher than in the controls. Higher parathyroid hormone concentrations were observed in the control group compared to the DL group at parturition. Pyridinoline concentrations did not differ between groups on d 1, and therefore it is assumed that the intensity of calcium transfer from the bones to the bloodstream was not affected by the increased daylight exposure. The measured plasma metabolites (β-hydroxybutyrate, fatty acids and glucose) as well as milk yield were not affected by the increased daylight exposure. In conclusion, the increased daylight exposure before parturition increased 25-VitD and 1,25-VitD concentrations, preventing a considerable decline of total calcium concentrations around parturition.

Parathyroid hormone and the regulation of renal tubular calcium transport.

Renal tubule excretion of calcium is carefully and complexly regulated. At the center of this process, parathyroid hormone (PTH) is the chief regulator of renal calcium reabsorption. This review outlines our current understanding of PTH's effects on renal tubular calcium transport, focusing on the more recent discoveries beyond its direct regulation of epithelial Ca channel transient receptor potential vanilloid 5 (TRPV5) and looking at its interaction with sodium transport and the hormones fibroblast growth factor 23 (FGF23) and klotho. PTH affects not only TRPV5 directly but other intracellular calcium transport proteins as well. In addition, PTH alters sodium transport that indirectly leads to enhanced TRPV5 activity. FGF23 and klotho have their own effects on TRPV5 as well as effects on PTH secretion. These discoveries and the interactions between these hormones have direct effects on our understanding of nephrolithiasis and have implications in the development of bone mineral disease in chronic kidney disease and may provide future treatment options.

Syndrome of inappropriate anti-diuresis induces volume-dependent hypercalciuria.

Chronic mild hyponatremia has been associated with bone demineralization of unknown mechanisms. During chronic hyponatremia, arginine-vasopressin secretion can result from hypovolemia or from syndrome of inappropriate anti-diuresis (SIAD) that leads to a slightly volemic expansion. Since volemia determines renal calcium excretion and balance, we evaluated calcium homeostasis in patients with chronic hyponatremia, related to SIAD or to hypovolemia. We retrospectively included all patients referred to our Department between May 2006 and May 2014 for hyponatremia, resulting from SIAD or chronic hypovolemia. None had edema, cirrhosis, cardiac, or renal insufficiency. Exploration included estimation of volemia, extracellular fluid volume (ECFV) measurement with inulin, and calcium homeostasis. In total, the SIAD and hypovolemic groups included 22 and 7 patients, respectively. The SIAD group exhibited signs of increased volemia: higher glomerular filtration rate, higher fractional excretion of uric acid, and lower plasma renin. ECFV exceeded that of the hypovolemic group and was above usual values. There was no difference between the two groups regarding plasma calcium, PTH, and vitamin D. However, in the SIAD group, calciuria was higher than in the hypovolemic group, reaching levels of hypercalciuria. Furthermore, there was a positive correlation between calciuria and markers of volemia. Our results show that SIAD results in a volemic expansion tendency that is associated with a decrease in renal calcium reabsorption and thus hypercalciuria, whereas in the hypovolemic group, calciuria was not increased. Therefore, renal loss of calcium and bone demineralization in SIAD patients could be partly induced by volemic expansion.

SaRNA-mediated activation of TRPV5 reduces renal calcium oxalate deposition in rat via decreasing urinary calcium excretion.

Hypercalciuria is a main risk factor for kidneystone formation. TRPV5is the gatekeeper protein for mediating calcium transport and reabsorption in the kidney. In the present study, we tested the effect of TRPV5 activation with small activating RNA (saRNA), which could induce gene expression by targeting the promoter of the gene, on ethylene glycol (EG)-induced calcium oxalate (CaOx) crystals formation in rat kidney. Five pairs of RNA activation sequences targeting the promoter of rat TRPV5 were designed and synthesized. The synthesized saRNA with the strongest activating effect was selected, and transcellular calcium transportation was tested by Fura-2 analysis. Subsequently, Sprague-Dawley rats were equally divided into three groups and fed with water, 1% EG for 28days after injecting the negative control saRNA, 1% EG for 28days after injecting the selected TRPV5-saRNA, respectively. The CaOx crystal formation and the 24-h urine components were assessed. In vitro study, saRNA ds-320 could significantly activate the expression of TRPV5 and transcellular calcium transportation. In vivo study, after 28days treatment of EG, rats pre-infected with saRNA ds-320 had lower urinary calcium excretion and renal CaOx crystals formation as compared to that pre-infected with negative control saRNA. Activation of TRVP5 with saRNA ds-320 could inhibit EG-induced calcium oxalate crystals formation via promoting urine calcium reabsorption and decreasing urine calcium excretion in rats.

Zoledronic acid and bone cellular respiration

Phosphorescence O2 analyzer was used to measure calvarial bone cellular respiration (cellular mitochondrial O2 consumption) in Taylor Outbred mice in the presence and absence of zoledronic acid. This potent bisphosphonate inhibits osteoclast-mediated calcium resorption, and its effects on bone respiration have not been previously investigated. The change of O2 concentration with time was measured in closed vials containing phosphate-buffered saline (PBS), 5mM glucose and 5-25mg calvarial bone fragments, and it was complex for t=0-30h. Cyanide (specific inhibitor of cytochrome oxidase) halted O2 consumption, confirming the oxidation occurred in the respiratory chain. Initial rate of respiration was estimated from the zero-order plots d[O2]/dt for t=0-4h. For untreated specimens, the rate (mean±SD) was 2.0±1.2µM O2h-1mg-1 (n=6). This value was 7-10 times lower than that of other murine organs, but similar to that reported for rat and Guinea pig calvaria (averaging, 2.7nmolO2h-1mg-1). The corresponding rate in the presence of 10-100µM zoledronic acid was 2.7±0.7µM O2h-1mg-1 (n=11), p=0.216. The first-order plots ln ([O2] t ÷[O2] t=0) versus time for t=0-30h were also used to compare treated and untreated specimens. The rate (h-1mg-1103) for specimens incubated in PBS without glucose was 1.3±0.6 (n=3, p=0.007), in PBS+glucose it was 10.7±6.9 (n=10), in PBS+glucose+10µM zoledronic acid it was 12.1±6.7 (n=10, p=0.579), in PBS+glucose+20µM zoledronic acid it was 12.9±3.3 (n=9, p=0.356), and in PBS+glucose+100µM zoledronic acid it was 13.7±7.7 (n=9, p=0.447). Thus, exposure to high-doses of zoledronic acid over several hours imposed a statistically insignificant increase in calvarial bone cellular respiration.

Abstract 3931: Sodium-calcium exchanger-1 regulates the epithelial phenotype and is lost in renal cancers

Abstract Epithelial cells that line the renal tubules possess apico-basal polarity and a defined cellular structure maintained by junctional proteins. The underlying mesenchymal cells are fibroblastic with a non-uniform cell structure and secrete extracellular matrix proteins. Mesenchymal to epithelial transition (MET) and epithelial to mesenchymal transition (EMT) are required for renal tubule formation during kidney development. Failure of renal mesenchymal cells to undergo epithelial transformation leads to the initiation of Wilms tumor, the most frequently occurring renal cancer in children. Conversely, aberrant mesenchymal transformation of tubular epithelial cells in the nephron contributes to the development of renal cell carcinoma, which constitutes 85-90% of the adult tubular malignancies. The role of calcium regulators in governing EMT and MET is becoming evident. Sodium calcium exchanger 1 (NCX1), located on the basolateral surface of tubular epithelial cells, is the principal calcium regulator that mediates calcium reabsorption in these cells. NCX1 mediates the extrusion of one calcium ion and the influx of three sodium ions in one exchange movement. We demonstrated earlier that NCX1 regulates epithelial cell motility. However, the role of NCX1 in EMT was undetermined. We observed that knockdown of NCX1 in renal epithelial cells (MDCK) induced fibroblastic morphology, and increased permeability to rhodamine indicative of leaky cell-cell junctions. Electron micrographs of these cells displayed increased inter-cellular junctional distance also suggesting that NCX1 knockdown altered junctions between adjacent cells. Cells with NCX1 knockdown showed loss of apico-basal polarity in three-dimensional cultures accompanied by expression of mesenchymal markers. Furthermore, NCX1 knockdown cells were capable of anchorage independent growth suggesting that these cells had acquired tumorigenic potential. Interestingly, we show for the first time that NCX1 mRNA and protein expression is greatly reduced in both Wilms tumor and renal cell carcinoma demonstrating a direct correlation between NCX1 expression and the epithelial phenotype. Mechanistically, we provide evidence that NCX1 interacts with and anchors E-cadherin, a classical adhesion molecule, to the cell surface independent of NCX1 ion transport activity. MDCK cells with NCX1 knockdown exhibited β-catenin nuclear localization and enhanced transcriptional activity. Taken together, knockdown of NCX1 in MDCK cells induces mesenchymal transition by destabilization of E-cadherin and induction of β-catenin transcriptional activity. Citation Format: Sona Lakshme Balasubramaniam, Anilkumar Gopalakrishnapillai, Nicholas J. Petrelli, Sonali P. Barwe. Sodium-calcium exchanger-1 regulates the epithelial phenotype and is lost in renal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3931. doi:10.1158/1538-7445.AM2017-3931

Interactions between calcium intake and polymorphisms in genes essential for calcium reabsorption and risk of colorectal neoplasia in a two-phase study.

The SLC8A1 (solute carrier family 8, member 1) gene, encoding Na+ /Ca2+ exchanger, is essential in regulating calcium reabsorption and homeostasis. Calcium homeostasis plays a key role in cell proliferation and apoptosis. We hypothesized that polymorphisms in five calcium-regulating genes (SLC8A1, ATP2B1, CALB1, CALB2, and CABP1) interact with calcium intake in relation to the risk of colorectal neoplasia. A two-phase (discovery and replication) study was conducted within the Tennessee Colorectal Polyp Study, including a total of 1275 cases and 2811 controls. In Phase I, we identified six out of 135 SNPs that significantly interacted with calcium intake in relation to adenoma risk. In Phase II, the calcium intake by rs4952490 (SLC8A1) interaction was replicated (Pinteraction = 0.048). We found an inverse association between calcium intake (1000-2000 mg/day) and colorectal adenomas, particularly for multiple/advanced adenomas, among the G-allele carriers but not among homozygous carriers of the common variant (A) in rs4952490. In the joint analysis of SLC8A1, KCNJ1, and SLC12A1 SNPs, carriers of variant alleles in at least two genes and with calcium intake above the DRI (1000 mg/day) were approximately 30-57% less likely to have adenomas than those whose calcium intake was below the DRI. The association was stronger for multiple/advanced adenomas. No association was found among those who did not carry any variant alleles in these genes when calcium intake was below 2500 mg/day. These findings, if confirmed, may provide a new avenue for the personalized prevention of colorectal adenoma and cancer.

Claudin Loss-of-Function Disrupts Tight Junctions and Impairs Amelogenesis.

Claudins are a family of proteins that forms paracellular barriers and pores determining tight junctions (TJ) permeability. Claudin-16 and -19 are pore forming TJ proteins allowing calcium and magnesium reabsorption in the thick ascending limb of Henle's loop (TAL). Loss-of-function mutations in the encoding genes, initially identified to cause Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC), were recently shown to be also involved in Amelogenesis Imperfecta (AI). In addition, both claudins were expressed in the murine tooth germ and Claudin-16 knockout (KO) mice displayed abnormal enamel formation. Claudin-3, an ubiquitous claudin expressed in epithelia including kidney, acts as a barrier-forming tight junction protein. We determined that, similarly to claudin-16 and claudin-19, claudin-3 was expressed in the tooth germ, more precisely in the TJ located at the apical end of secretory ameloblasts. The observation of Claudin-3 KO teeth revealed enamel defects associated to impaired TJ structure at the secretory ends of ameloblasts and accumulation of matrix proteins in the forming enamel. Thus, claudin-3 protein loss-of-function disturbs amelogenesis similarly to claudin-16 loss-of-function, highlighting the importance of claudin proteins for the TJ structure. These findings unravel that loss-of-function of either pore or barrier-forming TJ proteins leads to enamel defects. Hence, the major structural function of claudin proteins appears essential for amelogenesis.

Tradeoff-in-the-Nephron: A Theory to Explain the Primacy of Phosphate in the Pathogenesis of Secondary Hyperparathyroidism.

Chronic kidney disease (CKD) causes secondary hyperparathyroidism (SHPT). The cardinal features of SHPT are persistence of normocalcemia as CKD progresses and dependence of the parathyroid hormone concentration ([PTH]) on phosphate influx (IP). The tradeoff-in-the-nephron hypothesis integrates these features. It states that as the glomerular filtration rate (GFR) falls, the phosphate concentration ([P]CDN) rises in the cortical distal nephron, the calcium concentration ([Ca]CDN) in that segment falls, and [PTH] rises to maintain normal calcium reabsorption per volume of filtrate (TRCa/GFR). In a clinical study, we set GFR equal to creatinine clearance (Ccr) and IP equal to the urinary excretion rate of phosphorus (EP). We employed EP/Ccr as a surrogate for [P]CDN. We showed that TRCa/Ccr was high in patients with primary hyperparathyroidism (PHPT) and normal in those with SHPT despite comparably increased [PTH] in each group. In subjects with SHPT, we examined regressions of [PTH] on EP/Ccr before and after treatment with sevelamer carbonate or a placebo. All regressions were significant, and ∆[PTH] correlated with ∆EP/Ccr in each treatment cohort. We concluded that [P]CDN determines [PTH] in CKD. This inference explains the cardinal features of SHPT, much of the evidence on which other pathogenic theories are based, and many ancillary observations.

Interferon-γ Released by Activated CD8+ T Lymphocytes Impairs the Calcium Resorption Potential of Osteoclasts in Calcified Human Aortic Valves

In calcific aortic valve disease (CAVD), activated T lymphocytes localize with osteoclast regions; however, the functional consequences of this association remain unknown. We hypothesized that CD8+ T cells modulate calcification in CAVD. CAVD valves (n=52) dissected into noncalcified and calcified portions were subjected to mRNA extraction, real-time quantitative PCR, enzyme-linked immunosorbent assay, and immunohistochemical analyses. Compared with noncalcified portions, calcified regions exhibited elevated transcripts for CD8, interferon (IFN)-γ, CXCL9, Perforin 1, Granzyme B, and heat shock protein 60. Osteoclast-associated receptor activator of NK-κB ligand (RANKL), tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated receptor increased significantly. The stimulation of tissue with phorbol-12-myristate-13-acetate and ionomycin, recapitulating CAVD microenvironment, resulted in IFN-γ release. Real-time quantitative PCR detected mRNAs for CD8+ T-cell activation (Perforin 1, Granzyme B). In stimulated versus unstimulated organoid cultures, elevated IFN-γ reduced the mRNAs encoding for RANKL, TRAP, and Cathepsin K. Molecular imaging showed increased calcium signal intensity in stimulated versus unstimulated parts. CD14+ monocytes treated either with recombinant human IFN-γ or with conditioned media-derived IFN-γ exhibited low levels of Cathepsin K, TRAP, RANK, and tumor necrosis factor receptor-associated factor 6 mRNAs, whereas concentrations of the T-cell co-activators CD80 and CD86 increased in parallel with reduced osteoclast resorptive function, effects abrogated by neutralizing anti-IFN-γ antibodies. CD8+ cell-derived IFN-γ suppresses osteoclast function and may thus favor calcification in CAVD.