Interrelated role of Klotho and calcium-sensing receptor in parathyroid hormone synthesis and parathyroid hyperplasia.

The pathogenesis of parathyroid gland hyperplasia is poorly understood, and a better understanding is essential if there is to be improvement over the current strategies for prevention and treatment of secondary hyperparathyroidism. Here we investigate the specific role of Klotho expressed in the parathyroid glands (PTGs) in mediating parathyroid hormone (PTH) and serum calcium homeostasis, as well as the potential interaction between calcium-sensing receptor (CaSR) and Klotho. We generated mouse strains with PTG-specific deletion of Klotho and CaSR and dual deletion of both genes. We show that ablating CaSR in the PTGs increases PTH synthesis, that Klotho has a pivotal role in suppressing PTH in the absence of CaSR, and that CaSR together with Klotho regulates PTH biosynthesis and PTG growth. We utilized the tdTomato gene in our mice to visualize and collect PTGs to reveal an inhibitory function of Klotho on PTG cell proliferation. Chronic hypocalcemia and ex vivo PTG culture demonstrated an independent role for Klotho in mediating PTH secretion. Moreover, we identify an interaction between PTG-expressed CaSR and Klotho. These findings reveal essential and interrelated functions for CaSR and Klotho during parathyroid hyperplasia.

Calcium-Sensing Receptor Autoantibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1: Epitopes, Specificity, Functional Affinity, IgG Subclass, and Effects on Receptor Activity.

A major manifestation of autoimmune polyendocrine syndrome type 1 (APS1) is hypoparathyroidism, which is suggested to result from aberrant immune responses against the parathyroid glands. The calcium-sensing receptor (CaSR), which plays a pivotal role in maintaining calcium homeostasis by sensing blood calcium levels and regulating release of parathyroid hormone (PTH), is an autoantibody target in APS1. In this study, the aim was to characterize the binding sites, specificity, functional affinity, IgG subclass, and functional effects of CaSR autoantibodies using phage-display technology, ELISA, and bioassays. The results indicated that CaSR autoantibody binding sites were at aa 41-69, 114-126, 171-195, and 260-340 in the extracellular domain of the receptor. Autoantibodies against CaSR epitopes 41-69, 171-195, and 260-340 were exclusively of the IgG1 subclass. Autoantibody responses against CaSR epitope 114-126 were predominantly of the IgG1 with a minority of the IgG3 subclass. Only autoantibodies recognizing CaSR epitopes 114-126 and 171-195 affected receptor activity; inositol-phosphate accumulation was increased significantly in HEK293-CaSR cells, and PTH secretion from PTH-C1 cells was reduced significantly when either were incubated with purified Ab and Ca2+ compared with Ca2+ alone. In conclusion, although the majority of APS1 patients do not have CaSR-stimulating autoantibodies, the hypoparathyroid state in a small minority of patients is the result of functional suppression of the parathyroid glands.

Immunosuppressive therapy of autoimmune hypoparathyroidism in a patient with activating autoantibodies against the calcium-sensing receptor.

CONTEXT: Activating antibodies directed at the extracellular calcium-sensing receptor (CaSR) have been described in autoimmune hypoparathyroidism in the setting of isolated hypoparathyroidism or autoimmune polyglandular syndrome type 1. MATERIALS AND METHODS: A 34-year-old female presented with hypocalcaemia (6.0 mg/dL) and hypomagnesaemia (1.1 mg/dL) accompanied by low serum PTH (2.4 pg/mL) as well as urinary calcium and magnesium wasting. She was diagnosed with hypoparathyroidism, which was refractory to standard therapy. She was started on 60 mg prednisone and 150 mg azathioprine treatment daily on suspicion of an autoimmune aetiology. The patient was tested for CaSR antibodies. RESULTS: The patient was positive for CaSR antibodies of the IgG1 subtype, which stimulated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and inositol phosphate (IP) accumulation. Post-treatment with prednisone and azathioprine, her serum calcium and magnesium normalized, as did her CaSR antibody titre and antibody-mediated stimulation of ERK1/2 phosphorylation and IP accumulation. CONCLUSION: This is the first demonstration of CaSR antibody-mediated hypoparathyroidism responsive to immunosuppressive therapy, adding to the evidence that autoimmune hypoparathyroidism can be, in some cases, reversible and not the result of autoimmune parathyroid destruction.

François Leuret: the last moral therapist.

By the 1840s French psychiatrists had abandoned Moral Treatment as an individual psychological therapy, as opposed to an institutional practice. One advocate of Moral Treatment, however, would not go along with this movement. In three books and several papers published between 1834 and 1846, François Leuret (1797-1851) advocated aggressive psychological treatment. Recent commentators have understandably concentrated on the controversies surrounding Leuret's practices. What such an approach has failed to make clear, however, is that Leuret had a complex, systematic psychological theory supporting his clinical judgements. In addition to reviewing the controversies that surrounded Leuret, this paper spells out Leuret's psychological theory and shows how he used this theory to think about the individual psychotherapy he provided for his patients.

The calcium-sensing receptor: A promising target for prevention of colorectal cancer.

The inverse correlation between dietary calcium intake and the risk of colorectal cancer (CRC) is well known, but poorly understood. Expression of the calcium-sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is downregulated in CRC leading us to hypothesize that the CaSR has tumor suppressive roles in the colon. The aim of this study was to understand whether restoration of CaSR expression could reduce the malignant phenotype in CRC. In human colorectal tumors, expression of the CaSR negatively correlated with proliferation markers whereas loss of CaSR correlated with poor tumor differentiation and reduced apoptotic potential. In vivo, dearth of CaSR significantly increased expression of proliferation markers and decreased levels of differentiation and apoptotic markers in the colons of CaSR/PTH double knock-out mice confirming the tumor suppressive functions of CaSR. In vitro CRC cells stably overexpressing wild-type CaSR showed significant reduction in proliferation, as well as increased differentiation and apoptotic potential. The positive allosteric modulator of CaSR, NPS R-568 further enhanced these effects, whereas treatment with the negative allosteric modulator, NPS 2143 inhibited these functions. Interestingly, the dominant-negative mutant (R185Q) was able to abrogate these effects. Our results demonstrate a critical tumor suppressive role of CaSR in the colon. Restoration of CaSR expression and function is linked to regulation of the balance between proliferation, differentiation, and apoptosis and provides a rationale for novel strategies in CRC therapy.

Meta-analysis of genome-wide association studies identifies six new Loci for serum calcium concentrations.

Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤ 21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis.

Identification of an L-phenylalanine binding site enhancing the cooperative responses of the calcium-sensing receptor to calcium.

Functional positive cooperative activation of the extracellular calcium ([Ca(2+)]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca(2+)]o or amino acids elicits intracellular Ca(2+) ([Ca(2+)]i) oscillations. Here, we report the central role of predicted Ca(2+)-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca(2+)-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca(2+)]o. Next, we identify an adjacent L-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca(2+)]o and L-Phe in eliciting CaSR-mediated [Ca(2+)]i oscillations. The heterocommunication between Ca(2+) and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca(2+)]o signaling by positively impacting multiple [Ca(2+)]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca(2+)]o and amino acids into intracellular signaling events.

Direct determination of multiple ligand interactions with the extracellular domain of the calcium-sensing receptor.

Numerous in vivo functional studies have indicated that the dimeric extracellular domain (ECD) of the CaSR plays a crucial role in regulating Ca(2+) homeostasis by sensing Ca(2+) and l-Phe. However, direct interaction of Ca(2+) and Phe with the ECD of the receptor and the resultant impact on its structure and associated conformational changes have been hampered by the large size of the ECD, its high degree of glycosylation, and the lack of biophysical methods to monitor weak interactions in solution. In the present study, we purified the glycosylated extracellular domain of calcium-sensing receptor (CaSR) (ECD) (residues 20-612), containing either complex or high mannose N-glycan structures depending on the host cell line employed for recombinant expression. Both glycosylated forms of the CaSR ECD were purified as dimers and exhibit similar secondary structures with ∼ 50% α-helix, ∼ 20% ß-sheet content, and a well buried Trp environment. Using various spectroscopic methods, we have shown that both protein variants bind Ca(2+) with a Kd of 3.0-5.0 mm. The local conformational changes of the proteins induced by their interactions with Ca(2+) were visualized by NMR with specific (15)N Phe-labeled forms of the ECD. Saturation transfer difference NMR approaches demonstrated for the first time a direct interaction between the CaSR ECD and l-Phe. We further demonstrated that l-Phe increases the binding affinity of the CaSR ECD for Ca(2+). Our findings provide new insights into the mechanisms by which Ca(2+) and amino acids regulate the CaSR and may pave the way for exploration of the structural properties of CaSR and other members of family C of the GPCR superfamily.

Extracellular calcium modulates actions of orthosteric and allosteric ligands on metabotropic glutamate receptor 1α.

Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca(2+). However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca(2+)-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca(2+)-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca(2+) enhanced mGluR1α-mediated intracellular Ca(2+) responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca(2+) diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca(2+), respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca(2+) binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca(2+). These studies reveal that binding of extracellular Ca(2+) to the predicted Ca(2+)-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α.

The calcium-sensing receptor suppresses epithelial-to-mesenchymal transition and stem cell- like phenotype in the colon.

BACKGROUND: The calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown. METHODS AND RESULTS: In a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29(CaSR)) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29(EMP)). The HT29(CaSR) cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/ß-catenin pathway as measured by a decrease in nuclear translocation of ß-catenin and transcriptional regulation of genes like GSK-3ß and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29(CaSR) cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype. CONCLUSIONS: The results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.

Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites.

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that has an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium (Ca(2+))-binding sites. To elucidate the structure-function relationships of the VFTD, we investigated 294 unrelated probands with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT) or autosomal dominant hypocalcaemic hypercalciuria (ADHH) for CaSR mutations and performed in vitro functional expression studies and three-dimensional modelling of mutations involving the VFTD. A total of 70 different CaSR mutations were identified: 35 in FHH, 10 in NSHPT and 25 in ADHH patients. Furthermore, a CaSR variant (Glu250Lys) was identified in FHH and ADHH probands and demonstrated to represent a functionally neutral polymorphism. NSHPT was associated with a large proportion of truncating CaSR mutations that occurred in the homozygous or compound heterozygous state. Thirty-four VFTD missense mutations were identified, and 18 mutations were located within 10 Å of one or more of the predicted Ca(2+)-binding sites, particularly at the VFTD cleft, which is the principal site of Ca(2+) binding. Mutations of residues 173 and 221, which are located at the entrance to the VFTD cleft binding site, were associated with both receptor activation (Leu173Phe and Pro221Leu) and inactivation (Leu173Pro and Pro221Gln), thereby highlighting the importance of these residues for entry and binding of Ca(2+) by the CaSR. Thus, these studies of disease-associated CaSR mutations have further elucidated the role of the VFTD cleft region in Ca(2+) binding and the function of the CaSR.

The empirical foundations of telemedicine interventions for chronic disease management.

The telemedicine intervention in chronic disease management promises to involve patients in their own care, provides continuous monitoring by their healthcare providers, identifies early symptoms, and responds promptly to exacerbations in their illnesses. This review set out to establish the evidence from the available literature on the impact of telemedicine for the management of three chronic diseases: congestive heart failure, stroke, and chronic obstructive pulmonary disease. By design, the review focuses on a limited set of representative chronic diseases because of their current and increasing importance relative to their prevalence, associated morbidity, mortality, and cost. Furthermore, these three diseases are amenable to timely interventions and secondary prevention through telemonitoring. The preponderance of evidence from studies using rigorous research methods points to beneficial results from telemonitoring in its various manifestations, albeit with a few exceptions. Generally, the benefits include reductions in use of service: hospital admissions/re-admissions, length of hospital stay, and emergency department visits typically declined. It is important that there often were reductions in mortality. Few studies reported neutral or mixed findings.

CaSR-mediated interactions between calcium and magnesium homeostasis in mice.

Calcium (Ca) and magnesium (Mg) homeostasis are interrelated and share common regulatory hormones, including parathyroid hormone (PTH) and vitamin D. However, the role of the calcium-sensing receptor (CaSR) in Mg homeostasis in vivo is not well understood. We sought to investigate the interactions between Mg and Ca homeostasis using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR) (double knockout, DKO). Serum Mg is lower in PTH KO and DKO mice than in WT mice on standard chow, whereas supplemental dietary Ca leads to equivalent Mg levels for all three genotypes. Mg loading increases serum Mg in all genotypes; however, the increase in serum Mg is most pronounced in the DKO mice. Serum Ca is increased with Mg loading in the PTH KO and DKO mice but not in the WT mice. Here, too, the hypercalcemia is much greater in the DKO mice. Serum and especially urinary phosphate are reduced during Mg loading, which is likely due to intestinal chelation of phosphate by Mg. Mg loading decreases serum PTH in WT mice and increases serum calcitonin in both WT and PTH KO mice but not DKO mice. Furthermore, Mg loading elevates serum 1,25-dihydroxyvitamin D in all genotypes, with greater effects in PTH KO and DKO mice, possibly due to reduced levels of serum phosphorus and FGF23. These hormonal responses to Mg loading and the CaSR's role in regulating renal function may help to explain changes in serum Mg and Ca found during Mg loading.

Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo.

Calcium and phosphorus homeostasis are highly interrelated and share common regulatory hormones, including FGF23. However, little is known about calcium's role in the regulation of FGF23. We sought to investigate the regulatory roles of calcium and phosphorus in FGF23 production using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR; PTH-CaSR DKO). In wild-type, PTH KO, and PTH-CaSR DKO mice, elevation of either serum calcium or phosphorus by intraperitoneal injection increased serum FGF23 levels. In PTH KO and PTH-CaSR DKO mice, however, increases in serum phosphorus by dietary manipulation were accompanied by severe hypocalcemia, which appeared to blunt stimulation of FGF23 release. Increases in dietary phosphorus in PTH-CaSR DKO mice markedly decreased serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] despite no change in FGF23, suggesting direct regulation of 1,25(OH)(2)D(3) synthesis by serum phosphorus. Calcium-mediated increases in serum FGF23 required a threshold level of serum phosphorus of about 5 mg/dl. Analogously, phosphorus-elicited increases in FGF23 were markedly blunted if serum calcium was less than 8 mg/dl. The best correlation between calcium and phosphorus and serum FGF23 was found between FGF23 and the calcium × phosphorus product. Since calcium stimulated FGF23 production in the PTH-CaSR DKO mice, this effect cannot be mediated by the full-length CaSR. Thus the regulation of FGF23 by both calcium and phosphorus appears to be fundamentally important in coordinating the serum levels of both mineral ions and ensuring that the calcium × phosphorus product remains within a physiological range.

Acquired hypocalciuric hypercalcemia in a patient with CKD.

We present a case of an 82-year-old woman with elevated parathyroid hormone (PTH) levels, hypocalciuria, hypercalcemia, and stage 3 chronic kidney disease. Hypocalciuria initially was attributed to chronic kidney disease, and hypercalcemia was attributed to primary hyperparathyroidism. Subsequent laboratory studies showed autoantibodies in the patient's serum directed against the calcium-sensing receptor (CaSR). Functional testing in a CaSR-transfected human embryonic kidney-293 cell line showed that the patient's antibodies inhibited CaSR-mediated intracellular signaling that ordinarily would have been stimulated by extracellular calcium ions. Her serum calcium and PTH levels were normalized by treatment with the calcimimetic cinacalcet. We advise consideration of the presence of inhibitory autoantibodies directed at the CaSR in patients with hypercalcemic hyperparathyroidism and unexplained hypocalciuria or with confounding conditions affecting interpretation of urinary calcium measurement. A calcimimetic is an effective treatment for the hypercalcemia and elevated PTH levels in acquired hypocalciuric hypercalcemia caused by inhibitory anti-CaSR autoantibodies.

Parathyroid hormone ablation alters erythrocyte parameters that are rescued by calcium-sensing receptor gene deletion.

The mechanisms by which parathyroid hormone (PTH) produces anemia are unclear. Parathyroid hormone secretion is regulated by the extracellular Ca2+ -sensing receptor. We investigated the effects of ablating PTH on hematological indices and erythrocytes volume regulation in wild-type, PTH-null, and Ca2+ -sensing receptor-null/PTH-null mice. The erythrocyte parameters were measured in whole mouse blood, and volume regulatory systems were determined by plasma membrane K+ fluxes, and osmotic fragility was measured by hemoglobin determination at varying osmolarities. We observed that the absence of PTH significantly increases mean erythrocyte volume and reticulocyte counts, while decreasing erythrocyte counts, hemoglobin, hematocrit, and mean corpuscular hemoglobin concentration. These changes were accompanied by increases in erythrocyte cation content, a denser cell population, and increased K+ permeability, which were in part mediated by activation of the K+ /Cl- cotransporter and Gardos channel. In addition we observed that erythrocyte osmotic fragility in PTH-null compared with wild-type mice was enhanced. When Ca2+ -sensing receptor gene was deleted on the background of PTH-null mice, we observed that several of the alterations in erythrocyte parameters of PTH-null mice were largely rescued, particularly those related to erythrocyte volume, K+ fluxes and osmotic fragility, and became similar to those observed in wild-type mice. Our results demonstrate that Ca2+ -sensing receptor and parathyroid hormone are functionally coupled to maintain erythrocyte homeostasis.

Deficiency of the calcium-sensing receptor in the kidney causes parathyroid hormone-independent hypocalciuria.

Rare loss-of-function mutations in the calcium-sensing receptor (Casr) gene lead to decreased urinary calcium excretion in the context of parathyroid hormone (PTH)-dependent hypercalcemia, but the role of Casr in the kidney is unknown. Using animals expressing Cre recombinase driven by the Six2 promoter, we generated mice that appeared grossly normal but had undetectable levels of Casr mRNA and protein in the kidney. Baseline serum calcium, phosphorus, magnesium, and PTH levels were similar to control mice. When challenged with dietary calcium supplementation, however, these mice had significantly lower urinary calcium excretion than controls (urinary calcium to creatinine, 0.31±0.03 versus 0.63±0.14; P=0.001). Western blot analysis on whole-kidney lysates suggested an approximately four-fold increase in activated Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). In addition, experimental animals exhibited significant downregulation of Claudin14, a negative regulator of paracellular cation permeability in the thick ascending limb, and small but significant upregulation of Claudin16, a positive regulator of paracellular cation permeability. Taken together, these data suggest that renal Casr regulates calcium reabsorption in the thick ascending limb, independent of any change in PTH, by increasing the lumen-positive driving force for paracellular Ca(2+) transport.

The Ontario Telemedicine Network: a case report.

This article describes the evolution, current status, and future prospects of the Ontario Telemedicine Network (OTN). Started in the late 1990s (and formally established in 2006), OTN is a not-for-profit corporation primarily funded by the Government of Ontario, Canada, that aims to improve access to and quality of care throughout the Province. It covers a land mass larger than France and serves a population of just over 13 million, the vast majority of which live in a narrow strip close to the U.S. border. Telemedicine has been effective in reducing travel to usual sources of care, reducing hospital admissions, and improving efficiency and prompt access to care. The diffusion of telemedicine is accelerating in Ontario, and it is becoming an integral part of the health system.

Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca(2+) influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP(3) receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

The calcium-sensing receptor complements parathyroid hormone-induced bone turnover in discrete skeletal compartments in mice.

Although the calcium-sensing receptor (CaSR) and parathyroid hormone (PTH) may each exert skeletal effects, it is uncertain how CaSR and PTH interact at the level of bone in primary hyperparathyroidism (PHPT). Therefore, we simulated PHPT with 2 wk of continuous PTH infusion in adult mice with deletion of the PTH gene (Pth(-/-) mice) and with deletion of both PTH and CaSR genes (Pth(-/-)-Casr (-/-) mice) and compared skeletal phenotypes. PTH infusion in Pth(-/-) mice increased cortical bone turnover, augmented cortical porosity, and reduced cortical bone volume, femoral bone mineral density (BMD), and bone mineral content (BMC); these effects were markedly attenuated in PTH-infused Pth(-/-)-Casr(-/-) mice. In the absence of CaSR, the PTH-stimulated expression of receptor activator of nuclear factor-κB ligand and tartrate-resistant acid phosphatase and PTH-stimulated osteoclastogenesis was also reduced. In trabecular bone, PTH-induced increases in bone turnover, trabecular bone volume, and trabecular number were lower in Pth(-/-)-Casr(-/-) mice than in Pth(-/-) mice. PTH-stimulated genetic markers of osteoblast activity were also lower. These results are consistent with a role for CaSR in modulating both PTH-induced bone resorption and PTH-induced bone formation in discrete skeletal compartments.