Analysis of Calcium-Sensing Receptor Signaling Using Dual Luciferase Assays.

Luciferases catalyze a reaction that involves the emission of light, a phenomenon referred to as "bioluminescence". The calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), induces characteristic signaling pathways that stimulate extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ mobilization from the endoplasmic reticulum. ERK1/2 causes an activation of the serum response element (SRE), whereas Ca2+ causes an activation of the nuclear factor of activated T-cells response element (NFAT-RE). Transfection of cells with a vector containing a firefly luciferase reporter gene under the control of the SRE or NFAT-RE allows the monitoring of ERK1/2 activation and Ca2+ mobilization, respectively, by measuring luminescence. In a dual luciferase assay, firefly luminescence is normalized by co-transfecting an internal control vector, which contains a constitutively active promoter driving the expression of a second luciferase, namely, Renilla luciferase, whose activity can be quantified within the same sample. Here, a protocol for the analysis of CaSR signaling using dual luciferase assays in HEK293 cells is provided. The assays can, for example, be used to investigate functional consequences of mutations in the CaSR gene.

Exploring near-infrared autofluorescence properties in parathyroid tissue: an analysis of fresh and paraffin-embedded thyroidectomy specimens.

Significance: Near-infrared autofluorescence (NIRAF) utilizes the natural autofluorescence of parathyroid glands (PGs) to improve their identification during thyroid surgeries, reducing the risk of inadvertent removal and subsequent complications such as hypoparathyroidism. This study evaluates NIRAF's effectiveness in real-world surgical settings, highlighting its potential to enhance surgical outcomes and patient safety. Aim: We evaluate the effectiveness of NIRAF in detecting PGs during thyroidectomy and central neck dissection and investigate autofluorescence characteristics in both fresh and paraffin-embedded tissues. Approach: We included 101 patients diagnosed with papillary thyroid cancer who underwent surgeries in 2022 and 2023. We assessed NIRAF's ability to locate PGs, confirmed via parathyroid hormone assays, and involved both junior and senior surgeons. We measured the accuracy, speed, and agreement levels of each method and analyzed autofluorescence persistence and variation over 10 years, alongside the expression of calcium-sensing receptor (CaSR) and vitamin D. Results: NIRAF demonstrated a sensitivity of 89.5% and a negative predictive value of 89.1%. However, its specificity and positive predictive value (PPV) were 61.2% and 62.3%, respectively, which are considered lower. The kappa statistic indicated moderate to substantial agreement (kappa = 0.478; P < 0.001 ). Senior surgeons achieved high specificity (86.2%) and PPV (85.3%), with substantial agreement (kappa = 0.847; P < 0.001 ). In contrast, junior surgeons displayed the lowest kappa statistic among the groups, indicating minimal agreement (kappa = 0.381; P < 0.001 ). Common errors in NIRAF included interference from brown fat and eschar. In addition, paraffin-embedded samples retained stable autofluorescence over 10 years, showing no significant correlation with CaSR and vitamin D levels. Conclusions: NIRAF is useful for PG identification in thyroid and neck surgeries, enhancing efficiency and reducing inadvertent PG removals. The stability of autofluorescence in paraffin samples suggests its long-term viability, with false positives providing insights for further improvements in NIRAF technology.

Measuring IP3 Generation in Real-Time Using a NanoBiT Luminescence Biosensor.

G protein-coupled receptors that activate Gq/11 regulate a range of physiological processes including neurotransmission, energy homeostasis, blood pressure regulation, and calcium homeostasis. Activation of Gq/11-coupled receptors stimulates the generation of inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium release from the endoplasmic reticulum. This chapter describes an assay that uses a NanoBiT-IP3 luminescent biosensor to detect increases in IP3 in live cells. It describes how to perform these assays to assess signaling by the ghrelin receptor and the calcium-sensing receptor in HEK293 cells.

Monitoring Calcium-Sensing Receptor (CaSR)-Induced Intracellular Calcium Flux Using an Indo-1 Flow Cytometry Assay.

The calcium-sensing receptor (CaSR) has a critical role in maintaining serum calcium concentrations within the normal physiological range, and mutations in the receptor, or components of its signaling and trafficking pathway, cause disorders of calcium homeostasis. Inactivating mutations cause neonatal severe hyperparathyroidism or familial hypocalciuric hypercalcemia (FHH), while gain-of-function mutations cause autosomal dominant hypocalcemia (ADH). Characterizing the functional impact of mutations of the CaSR, and components of the CaSR-signaling pathway, is clinically important to enable correct diagnoses of FHH and ADH, optimize management, and prevent inappropriate parathyroidectomy or vitamin D supplementation. CaSR signals predominantly by activating the G-alpha subunit-11 to mobilize calcium release from intracellular stores. Thus, measurement of CaSR-induced intracellular calcium (Ca2+i) signaling is the gold standard method to investigate the pathogenicity of CaSR genetic variants. This protocol describes a method to assess CaSR-induced Ca2+I signaling using the Indo-1 calcium indicator dye and flow cytometry. This method has been used to assess multiple genetic variants in CaSR and components of its signaling and trafficking pathway in HEK293 cells.

Measuring Calcium-Sensing Receptor Agonist-Driven Insertional Signaling (ADIS) and Trafficking by TIRF Microscopy.

The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor. Deciphering these mechanisms is important to understand how mutations in the CaSR and components of its signaling and trafficking pathways cause human disorders of calcium homeostasis.This chapter describes a protocol to measure CaSR ADIS and endocytosis in parallel using total internal reflection fluorescence (TIRF) microscopy. This utilizes a mammalian expression construct comprising the full-length human CaSR with an N-terminal bungarotoxin minimal-binding site that can be labeled with commercially available fluorescent ligands to measure endocytosis, and a super-ecliptic pHluorin (SEP) to measure total cell surface expression and exocytic events. This protocol could easily be adapted to simultaneously assess forward trafficking and endocytosis of other membrane proteins by TIRF microscopy.

Mechanisms of Xuefu Zhuyu Tang in the Treatment of Diabetic Erectile Dysfunction in Rats Through the Regulation of Vascular Endothelial Function by CaSR/PLC/PKC and MEK/ERK/RSK Pathways.

Xuefu zhuyu Tang (XFZYT) is a classic formula used for promoting blood circulation and resolving blood stasis in Traditional Chinese Medicine. Clinical data have indicated that XFZYT plays a significant therapeutic role in diabetes-induced erectile dysfunction (DIED) disease, but the underlying mechanism remains elusive. Male Sprague-Dawley (SD) rats were randomly categorized into normal, model, and treatment groups. The diabetic rat model was established via intraperitoneal injection of streptozotocin. DIED rats were screened using apomorphine, and the number of erections was measured after 8 weeks of XFZYT treatment. Serum nitric oxide (NO) and endothelin-1 levels as well as penile tissue structure alterations were assessed by hematoxylin-eosin staining and electron microscopy. CaSR/PLC/PKC and MEK/ERK/RSK pathway-related proteins in the penile tissue were detected by western blotting (WB) analysis and polymerase chain reaction (PCR). Compared with the blank group, the model group rats showed a significant decrease in weight and erectile function. The pathological damage in the penile tissues of the model rats was indicated by a significantly decreased serum NO level and an increased endothelin-1 content. After treatment with XFZYT, the protein expression of CaSR, PLCß1, PKCß, MEK1, ERK1, and RSK1 in the penile tissue was significantly increased. Overall, the treatment group showed significant improvements in the evaluated indexes. In conclusion, this study revealed that XFZYT improves erectile function in diabetic rats, and the underlying mechanism might be linked with the regulation of CaSR/PLC/PKC and related molecules of the MEK/ERK/RSK pathway, which promotes the vascular endothelial diastolic effect.

Comparative analysis of calcium-sensing receptor (CaSR) expression and function in normal and abnormal human sperm and spermatogenic cells.

The calcium-sensing receptor (CaSR) is a critical mediator of calcium homeostasis in various tissues. Its role in human reproduction, especially in sperm function and male fertility, remains not fully elucidated. This study investigates the expression patterns of CaSR in normal and abnormal sperm and spermatogenic cells and evaluates its potential effect on sperm motility and morphology. Using immunohistochemistry (IHC), quantitative PCR (qPCR), we assessed the expression levels of CaSR in normal sperm, spermatogonia, and cases of asthenozoospermia, oligozoospermia, and teratozoospermia. In vitro functional assays were performed to analyze the effects of CaSR modulation on sperm motility under varying conditions, including the presence of specific CaSR agonists and antagonists. Our study revealed distinct patterns of CaSR expression in normal sperm and spermatogonia compared with those in abnormal sperm samples, particularly in cases of asthenozoospermia, oligozoospermia, and teratozoospermia. A marked decrease in CaSR expression was evident in these abnormal samples, highlighting its significance in normal sperm functionality. Functional assays further elucidated the role of CaSR in sperm motility. Activation of CaSR through specific agonists enhanced sperm motility, while inhibition by antagonists led to reduced motility. Our findings suggest that CaSR plays a significant role in maintaining sperm functionality and that changes in its expression may be associated with male infertility. These insights into the molecular underpinnings of sperm physiology highlight CaSR as a potential therapeutic target for treating certain forms of male infertility.

Pea protein hydrolysate stimulates GLP-1 secretion in NCI-H716 cells via simultaneously activating the sensing receptors CaSR and PepT1.

Glucagon-like peptide-1 (GLP-1) plays a crucial role in regulating glucose homeostasis by stimulating insulin secretion and suppressing glucagon release. Our previous study observed that pea protein hydrolysate (PPH) exhibited the function of triggering GLP-1 secretion. However, the underlying mechanisms have not been revealed. Herein, the mechanisms of PPH-stimulated GLP-1 secretion were investigated in NCI-H716 cells. The PPH-induced GLP-1 secretion was reduced (p < 0.05) after adding the sensing receptor antagonists NPS-2143 and 4-AMBA, indicating that activation of both calcium-sensing receptor (CaSR) and peptide-transporter 1 (PepT1) was involved in PPH-triggered GLP-1 release. Moreover, the intracellular Ca2+ level increased by 2.01 times during the PPH-induced GLP-1 secretion. Similarly, the cAMP content also increased by 1.43 times after stimulation by PPH. The RT-qPCR results showed that PPH increased the gene expression of prohormone convertase 1/3 (PCSK-1) by 2.79-fold, which effectively promoted the conversion of proglucagon (GCG) to GLP-1. The specific pathway of PPH-induced GLP-1 secretion may involve both CaSR and PepT1 activation-induced Ca2+ influx and cAMP generation, which effectively enhanced the enzyme activity of prohormone convertase 1/3 (PCSK-1) and ultimately promoted GLP-1 secretion.

Targeting protein receptors and enzymes for precision management of urolithiasis: A comprehensive review.

Urolithiasis, characterized by the formation of solid crystalline structures within the urinary tract, presents a significant global health burden with high recurrence rates and limited treatment efficacy. Recent research has identified various protein receptors and enzymes implicated in the pathogenesis of urolithiasis, offering potential targets for therapeutic intervention. Protein receptors such as the calcium-sensing receptor and vasopressin V2 receptor play crucial roles in regulating urinary calcium excretion and water reabsorption, respectively, influencing stone formation. Additionally, modulation of receptors like the angiotensin II receptor and aldosterone receptor can impact renal function and electrolyte balance, contributing to stone prevention. Furthermore, enzymes such as urease inhibitors and xanthine oxidase inhibitors offer targeted approaches to prevent the formation of specific stone types. This review discusses the potential of targeting these receptors and enzymes for the treatment of urolithiasis, exploring associated drugs and their mechanisms of action. Despite promising avenues for personalized and precision medicine approaches, challenges such as the need for robust clinical evidence and ensuring cost-effectiveness must be addressed for the translation of these interventions into clinical practice. By overcoming these challenges, receptor-targeted therapies and enzyme inhibitors hold promise for revolutionizing the management of urolithiasis and reducing its global burden.

The calcium-sensing-receptor (CaSR) in adipocytes contributes to sex-differences in the susceptibility to high fat diet induced obesity and atherosclerosis.

BACKGROUND: Female mice are more resistant to obesogenic effects of a high-fat diet (HFD), compared to male mice. Although the underlying mechanisms are poorly understood, sex hormones seem to play an important role. Interestingly, the activity of the oestrogen receptor-α (ERα) is affected by the calcium-sensing-receptor (CaSR). Therefore, we investigated sex-differences upon diet-induced obesity and the role of adipocyte-specific CaSR herein. METHODS: Adipocyte-specific Casr deficient mice (AdipoqCre+Casrflox) and control mice (Casrflox) were injected with AAV8-PCSK9 to make them prone to develop atherosclerosis and fed an obesity-inducing diet for 12 weeks. FINDINGS: Female mice have lower visceral white adipose tissue (vWAT) mass compared to male mice, while this sex-difference is abolished upon adipocyte-specific Casr deficiency. Furthermore, while females showed elevated levels of inflammatory cytokines and CD3+CD8+ T cell accumulation in vWAT, compared to males, adipocyte-specific Casr deficiency abrogated this sex-phenotype and demonstrated an inhibition of inflammatory signalling pathways. The expression of Erα, as well as associated genes involved in adipocyte differentiation, was increased in female mice in a mostly adipocyte-specific Casr dependent manner. Interestingly, circulating lipid levels were reduced in female compared to male mice, which correlated with decreased atherosclerotic plaque formation. These systemic effects were abrogated upon adipocyte-specific Casr deficiency. INTERPRETATION: Our findings indicate that female mice show a more pronounced vWAT dysfunction compared to males upon obesity. This sex effect is abolished upon adipocyte-specific Casr deficiency. In contrast, females show diminished atherosclerotic plaque formation compared to males, an effect that was abrogated by adipocyte-specific Casr deficiency. FUNDING: This work was supported by a grant from the Interdisciplinary Center for Clinical Research within the faculty of Medicine at the RWTH Aachen University, by the Corona Foundation, by the Deutsche Forschungsgemeinschaft (DFG), the BMBF and Free State of Bavaria and the DZHK.

The Interaction of Calcium-Sensing Receptor with KIF11 Enhances Cisplatin Resistance in Lung Adenocarcinoma via BRCA1/cyclin B1 pathway.

Cisplatin (DDP) is commonly used in the treatment of non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD), and the primary cause for its clinical inefficacy is chemoresistance. Here, we aimed to investigate a novel mechanism of chemoresistance in LUAD cells, focusing on the calcium-sensing receptor (CaSR). In this study, high CaSR expression was detected in DDP-resistant LUAD cells, and elevated CaSR expression is strongly correlated with poor prognosis in LUAD patients receiving chemotherapy. LUAD cells with high CaSR expression exhibited decreased sensitivity to cisplatin, and the growth of DDP-resistant LUAD cells was inhibited by cisplatin treatment in combination with CaSR suppression, accompanied by changes in BRCA1 and cyclin B1 protein expression both in vitro and in vivo. Additionally, an interaction between CaSR and KIF11 was identified. Importantly, suppressing KIF11 resulted in decreased protein levels of BRCA1 and cyclin B1, enhancing the sensitivity of DDP-resistant LUAD cells to cisplatin with no obvious decrease in CaSR. Here, our findings established the critical role of CaSR in promoting cisplatin resistance in LUAD cells by modulating cyclin B1 and BRCA1 and identified KIF11 as a mediator, highlighting the potential therapeutic value of targeting CaSR to overcome chemoresistance in LUAD.

Calcium-sensing receptor regulates the angiogenic differentiation of LPS-treated human dental pulp cells via the phosphoinositide 3-kinase/Akt pathway in vitro.

AIM: The purpose of this study was to investigate the role of calcium-sensing receptor (CaSR) in the angiogenic differentiation of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). METHODOLOGY: The LPS-induced hDPCs were cultured in the medium with different combinations of CaSR agonist R568 and antagonist Calhex231. The cell proliferation, migration, and angiogenic capacity were measured by Cell Counting Kit-8 (CCK-8), scratch wound healing, and tube formation assays, respectively. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were conducted to determine the gene/protein expression of CaSR, inflammatory mediators, and angiogenic-associated markers. The activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) was assessed by western blot analysis. RESULTS: The cell proliferation was elevated in response to R568 or Calhex231 exposure, but an enhanced cell migration was only found in cultures supplemented with Calhex231. Furthermore, R568 was found to potentiate the formation of vessel-like structure, up-regulated the protein expression of tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF)-1; comparable influences were also observed in R568-stimulated cells in the presence of PI3K inhibitor LY294002. In contrast, Calhex231 obviously inhibited the tube formation and VEGF protein level, whereas promoted the production of IL-6, TNF-α, and eNOS; however, in the presence of LY294002, Calhex231 showed a significant promotion on the protein expression of CaSR, VEGF, and SDF-1. In addition, R568 exhibited a promotive action on the Akt phosphorylation, which can be reversed by LY294002. CONCLUSIONS: Our results demonstrated that CaSR can regulate the angiogenic differentiation of LPS-treated hDPCs with an involvement of the PI3K/Akt signalling pathway.

Novel osteogenic peptide from bovine bone collagen hydrolysate: Targeted screening, molecular mechanism, and stability analysis.

This study aimed to screen for a novel osteogenic peptide based on the calcium-sensing receptor (CaSR) and explore its molecular mechanism and gastrointestinal stability. In this study, a novel osteogenic peptide (Phe-Ser-Gly-Leu, FSGL) derived from bovine bone collagen hydrolysate was successfully screened by molecular docking and synthesised by solid phase peptide synthesis for further analysis. Cell experiments showed that FSGL significantly enhanced the osteogenic activity of MC3T3-E1 cells by acting on CaSR, including proliferation (152.53%), differentiation, and mineralization. Molecular docking and molecular dynamics further demonstrated that FSGL was a potential allosteric activator of CaSR, that turned on the activation switch of CaSR by closing the Venus flytrap (VFT) domain and driving the two protein chains in the VFT domain to easily form dimers. In addition, 96.03% of the novel osteogenic peptide FSGL was stable during gastrointestinal digestion. Therefore, FSGL showed substantial potential for enhancing the osteogenic activity of osteoblasts. This study provided new insights for the application of CaSR in the targeted screening of osteogenic peptides to improve bone health.

Analysis of Expression of the ANG1, CaSR and FAK Proteins in Uterine Fibroids.

Understanding the molecular factors involved in the development of uterine myomas may result in the use of pharmacological drugs instead of aggressive surgical treatment. ANG1, CaSR, and FAK were examined in myoma and peripheral tissue samples taken from women after myoma surgery and in normal uterine muscle tissue samples taken from the control group. Tests were performed using tissue microarray immunohistochemistry. No statistically significant differences in ANG1 expression between the tissue of the myoma, the periphery, and the normal uterine muscle tissue of the control group were recorded. The CaSR value was reduced in the myoma and peripheral tissue and normal in the group of women without myomas. FAK expression was also lower in the myoma and periphery compared to the healthy uterine myometrium. Calcium supplementation could have an effect on stopping the growth of myomas.

Glutathione-Based Photoaffinity Probe Identifies Caffeine as a Positive Allosteric Modulator of the Calcium-Sensing Receptor.

The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, and bone remodeling, in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione, cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we developed a glutathione-based photoaffinity probe, DAZ-G, that analyzes ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium, cinacalcet, and etelcalcetide have cooperative effects. An unexpected finding was that caffeine effectively competes with DAZ-G's conjugation to CaSR and acts as a positive allosteric modulator of CaSR. The effective concentration of caffeine for CaSR activation (<10 µM) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.

Stimulation of the calcium-sensing receptor induces relaxations through CGRP and NK1 receptor-mediated pathways in male rat mesenteric arteries.

Stimulation of the calcium-sensing receptor (CaSR) regulates vascular contractility, but cellular mechanisms involved remain unclear. This study investigated the role of perivascular sensory nerves in CaSR-induced relaxations of male rat mesenteric arteries. In fluorescence studies, colocalisation between synaptophysin, a synaptic vesicle marker, and the CaSR was present in the adventitial layer of arterial segments. Using wire myography, increasing external Ca2+ concentration ([Ca2+]o) from 1 to 10 mM induced vasorelaxations, previously shown to involve the CaSR, which were inhibited by pretreatment with capsaicin. [Ca2+]o-induced vasorelaxations were partially reduced by the calcitonin gene-related peptide (CGRP) receptor blockers, CGRP 8-37 and BIBN 4096, and the neurokinin 1 (NK1) receptor blocker L733,060. The inhibitory effect of CGRP 8-37 required a functional endothelium whereas the inhibitory action of L733,060 did not. Complete inhibition of [Ca2+]o-induced vasorelaxations occurred when CGRP 8-37 and L733,060 were applied together. [Ca2+]o-induced vasorelaxations in the presence of capsaicin were abolished by the ATP-dependent K+ channel (KATP) blocker PNU 37883, but unaffected by the endothelium nitric oxide synthase (eNOS) inhibitor L-NAME. We suggest that the CaSR on perivascular sensory nerves mediate relaxations in rat mesenteric arteries via endothelium-dependent and -independent mechanisms involving CGRP and NK1 receptor-activated NO production and KATP channels, respectively.

Ca2+-sensing receptor-TRP channel-mediated Ca2+ signaling: Functional diversity and pharmacological complexity.

The Ca2+-sensing receptor (CaSR) is a G-protein-coupled receptor activated by elevated concentrations of extracellular Ca2+, and was initially known for its regulation of parathyroid hormone (PTH) release. Ubiquitous expression of CaSR in different tissues and organs was later noted and CaSR participation in various physiological functions was demonstrated. Accumulating evidence has suggested that CaSR functionally interacts with transient receptor potential (TRP) channels, which are mostly non-selective cation channels involved in sensing temperature, pain and stress. This review describes the interactions of CaSR with TRP channels in diverse cell types to trigger a variety of biological responses. CaSR has been known to interact with different types of G proteins. Possible involvements of G proteins, other signaling and scaffolding protein intermediates in CaSR-TRP interaction are discussed. In addition, an attempt will be made to extend the current understanding of biased agonism of CaSR.

Phosphate as an adjunct to calcium in promoting coronary vascular calcification in chronic inflammatory states.

Bone releases calcium and phosphate in response to pro-inflammatory cytokine-mediated inflammation. The body develops impaired urinary excretion of phosphate with age and chronic inflammation given the reduction of the kidney protein Klotho, which is essential to phosphate excretion. Phosphate may also play a role in the development of the resistance of the parathyroid calcium-sensing receptor (CaSR) to circulating calcium thus contributing to calcium retention in the circulation. Phosphate can contribute to vascular smooth muscle dedifferentiation with manifestation of osteoblastogenesis and ultimately endovascular calcium phosphate precipitation. Thus phosphate, along with calcium, contributes to the calcification and inflammation of atherosclerotic plaques and the origin of these elements is likely the bone, which serves as storage for the majority of the body's supply of extracellular calcium and phosphate. Early cardiac evaluation of patients with chronic inflammation and attempts at up-regulating the parathyroid CaSR with calcimimetics or introducing earlier anti-resorptive treatment with bone active pharmacologic agents may serve to delay onset or reduce the quantity of atherosclerotic plaque calcification in these patients.

New insights into renal calcium-sensing receptor activation.

PURPOSE OF REVIEW: Activation of the calcium-sensing receptor (CASR) in the parathyroid gland suppresses the release of parathyroid hormone (PTH). Furthermore, activation of the renal CASR directly increases the urinary excretion of calcium, by inhibiting transepithelial calcium transport in the nephron. Gain-of-function mutations in the CASR gene lead to autosomal dominant hypocalcemia 1 (ADH1), with inappropriately low PTH levels and hypocalcemia, indicative of excessive activation of the parathyroid CASR. However, hypercalciuria is not always observed. The reason why the manifestation of hypercalciuria is not uniform among ADH1 patients is not well understood. RECENT FINDINGS: Direct activation of the CASR in the kidney has been cumbersome to study, and an indirect measure to effectively estimate the degree of CASR activation following chronic hypercalcemia or genetic gain-of-function CASR activation has been lacking. Studies have shown that expression of the pore-blocking claudin-14 is strongly stimulated by the CASR in a dose-dependent manner. This stimulatory effect is abolished after renal Casr ablation in hypercalcemic mice, suggesting that claudin-14 abundance may gauge renal CASR activation. Using this marker has led to unexpected discoveries regarding renal CASR activation. SUMMARY: These new studies have informed on renal CASR activation thresholds and the downstream CASR-regulated calcium transport mechanisms.