The Molecular Basis of Calcium and Phosphorus Inherited Metabolic Disorders.

Calcium (Ca) and Phosphorus (P) hold a leading part in many skeletal and extra-skeletal biological processes. Their tight normal range in serum mirrors their critical role in human well-being. The signalling "voyage" starts at Calcium Sensing Receptor (CaSR) localized on the surface of the parathyroid glands, which captures the "oscillations" of extracellular ionized Ca and transfers the signal downstream. Parathyroid hormone (PTH), Vitamin D, Fibroblast Growth Factor (FGF23) and other receptors or ion-transporters, work synergistically and establish a highly regulated signalling circuit between the bone, kidneys, and intestine to ensure the maintenance of Ca and P homeostasis. Any deviation from this well-orchestrated scheme may result in mild or severe pathologies expressed by biochemical and/or clinical features. Inherited disorders of Ca and P metabolism are rare. However, delayed diagnosis or misdiagnosis may cost patient's quality of life or even life expectancy. Unravelling the thread of the molecular pathways involving Ca and P signaling, we can better understand the link between genetic alterations and biochemical and/or clinical phenotypes and help in diagnosis and early therapeutic intervention.

Alzheimer's Disease Presenilin-1 Mutation Sensitizes Neurons to Impaired Autophagy Flux and Propofol Neurotoxicity: Role of Calcium Dysregulation.

BACKGROUND: Disruption of intracellular Ca2+ homeostasis and associated autophagy dysfunction contribute to neuropathology in Alzheimer's disease (AD). OBJECTIVE: To study the effects of propofol on cell viability via its effects on intracellular Ca2+ homeostasis, and the impact of autophagy, in a neuronal model of presenilin-mutated familial AD (FAD). METHODS: We treated PC12 cells, stably transfected with either mutated presenilin-1 (L286V) or wild type (WT) controls, with propofol at different doses and durations, in the presence or absence of extracellular Ca2+, antagonists of inositol trisphosphate receptors (InsP3R, xestospongin C) and/or ryanodine receptors (RYR, dantrolene), or an inhibitor of autophagy flux (Bafilomycin). We determined cell viability, cytosolic Ca2+ concentrations ([Ca2+]c), vATPase protein expression, and lysosomal acidification. RESULTS: The propofol dose- and time-dependently decreased cell viability significantly more in L286V than WT cells, especially at the pharmacological dose (>50µM), and together with bafilomycin (40 nM). Clinically used concentrations of propofol (<20µM) tended to increase cell viability. Propofol significantly increased [Ca2+]c more in L286V than in WT cells, which was associated with decrease of vATPase expression and localization to the lysosome. Both toxicity and increased Ca2+ levels were ameliorated by inhibiting InsP3R/RYR. However, the combined inhibition of both receptors paradoxically increased [Ca2+]c, by inducing Ca2+ influx from the extracellular space, causing greater cytotoxicity. CONCLUSION: Impairment in autophagy function acts to deteriorate cell death induced by propofol in FAD neuronal cells. Cell death is ameliorated by either RYR or InsP3R antagonists on their own, but not when both are co-administered.

Disordered myocardial Ca(2+) homeostasis results in substructural alterations that may promote occurrence of malignant arrhythmias.

We aimed to determine the impact of Ca(2+)-related disorders induced in intact animal hearts on ultrastructure of the cardiomyocytes prior to occurrence of severe arrhythmias. Three types of acute experiments were performed that are known to be accompanied by disturbances in Ca(2+) handling. Langedorff-perfused rat or guinea pig hearts subjected to K(+)-deficient perfusion to induce ventricular fibrillation (VF), burst atrial pacing to induce atrial fibrillation (AF) and open chest pig heart exposed to intramyocardial noradrenaline infusion to induce ventricular tachycardia (VT). Tissue samples for electron microscopic examination were taken during basal condition, prior and during occurrence of malignant arrhythmias. Cardiomyocyte alterations preceding occurrence of arrhythmias consisted of non-uniform sarcomere shortening, disruption of myofilaments and injury of mitochondria that most likely reflected cytosolic Ca(2+) disturbances and Ca(2+) overload. These disorders were linked with non-uniform pattern of neighboring cardiomyocytes and dissociation of adhesive junctions suggesting defects in cardiac cell-to-cell coupling. Our findings identified heterogeneously distributed high [Ca(2+)](i)-induced subcellular injury of the cardiomyocytes and their junctions as a common feature prior occurrence of VT, VF or AF. In conclusion, there is a link between Ca(2+)-related disorders in contractility and coupling of the cardiomyocytes pointing out a novel paradigm implicated in development of severe arrhythmias.

Roles of the calcium sensing receptor in digestive physiology and pathophysiology (review).

Calcium participates in most of the biological processes in the human body. The calcium sensing receptor (CaSR), as an important regulator of calcium homeostasis, is expressed in all of the organs of the digestive system. CaSR plays a key role in gastrointestinal physiological function and in the occurrence of digestive disease. For example, the inactivation or mutation of the CaSR gene usually leads to one of several disorders of calcium metabolism. High dietary Ca2+ may stimulate CaSR activation and could both inhibit tumor development and increase the chemotherapeutic sensitivity of cancer cells in colon cancer tissues. Further, CaSR has also been reported to have a potential role in the treatment for diarrheal diseases and the form of pancreatitis that is associated with carbonate stones. Therefore, CaSR is an important target for treating digestive diseases, and the calcimimetics (CaSR agonist) have been confirmed as practical, feasible and effective clinical therapies for hyperparathyroidism. This review intends to explore the role of CaSR in digestive physiology and pathophysiology as well as current treatments utilizing CaSR­based therapeutics.

Disturbances in calcium metabolism and cardiomyocyte necrosis: the role of calcitropic hormones.

A synchronized dyshomeostasis of extra- and intracellular Ca(2+), expressed as plasma ionized hypocalcemia and excessive intracellular Ca(2+) accumulation, respectively, represents a common pathophysiologic scenario that accompanies several diverse disorders. These include low-renin and salt-sensitive hypertension, primary aldosteronism and hyperparathyroidism, congestive heart failure, acute and chronic hyperadrenergic stressor states, high dietary Na(+), and low dietary Ca(2+) with hypovitaminosis D. Homeostatic responses are invoked to restore normal extracellular [Ca(2+)](o), including increased plasma levels of parathyroid hormone and 1,25(OH)(2)D(3). However, in cardiomyocytes these calcitropic hormones concurrently promote cytosolic free [Ca(2+)](i) and mitochondrial [Ca(2+)](m) overloading. The latter sets into motion organellar-based oxidative stress, in which the rate of reactive oxygen species generation overwhelms their detoxification by endogenous antioxidant defenses, including those related to intrinsically coupled increments in intracellular Zn(2+). In turn, the opening potential of the mitochondrial permeability transition pore increases, allowing for osmotic swelling and ensuing organellar degeneration. Collectively, these pathophysiologic events represent the major components to a mitochondriocentric signal-transducer-effector pathway to cardiomyocyte necrosis. From necrotic cells, there follows a spillage of intracellular contents, including troponins, and a subsequent wound healing response with reparative fibrosis or scarring. Taken together, the loss of terminally differentiated cardiomyocytes from this postmitotic organ and the ensuing replacement fibrosis each contribute to the adverse structural remodeling of myocardium and progressive nature of heart failure. In conclusion, hormone-induced ionized hypocalcemia and intracellular Ca(2+) overloading comprise a pathophysiologic cascade common to diverse disorders and that initiates a mitochondriocentric pathway to nonischemic cardiomyocyte necrosis.

Strio-pallido-dentate calcinosis: a diagnostic approach in adult patients.

Familial idiopathic bilateral strio-pallido-dentate calcinosis is a rare autosomal dominant disorder characterized by massive symmetric calcification, detectable by CT, into the globus pallidus and striatum, with or without the involvement of the dentate nucleus, thalamus and white matter in the absence of alterations of calcium metabolism. Clinically, it has been associated with movement and/or neuropsychiatric disorders with age at onset typically in the fourth or fifth decade. Other sporadic or familial diseases can be responsible for brain calcifications with a similar anatomic strio-pallidal or strio-pallido-dentate pattern and, a restricted number of them, for neurological symptoms with onset in adulthood. Moreover, physiological age-related basal ganglia calcifications are often incidentally found, although with a far different CT aspect, in elderly patients with movement disorders. Indentifying familial and idiopathic cases may offer the opportunity to study the molecular mechanisms underlying this minerals deposition.

Hyperglycemic nonketotic states and other metabolic imbalances.

Hemichorea and generalized chorea are well-recognized syndromes associated with nonketotic hyperglycemia. This condition usually occurs in older age, affects females more than men, and often heralds a new diagnosis of diabetes, usually type 2. It may resolve over days with treatment of the underlying hyperglycemia or persist for years. Magnetic resonance imaging is very characteristic, and shows T1 hyperdensity in the striatum. The underlying pathophysiology is not clear, but recent evidence suggests that the imaging may represent zinc, as opposed to calcium. Tetrabenazine has worked well when symptomatic treatment is required. Other rare causes of metabolic choreas include hypoparathyroid abnormalities, hypoglycemia, and hypernatremia.

Endoplasmic-reticulum calcium depletion and disease.

The endoplasmic reticulum (ER) as an intracellular Ca(2+) store not only sets up cytosolic Ca(2+) signals, but, among other functions, also assembles and folds newly synthesized proteins. Alterations in ER homeostasis, including severe Ca(2+) depletion, are an upstream event in the pathophysiology of many diseases. On the one hand, insufficient release of activator Ca(2+) may no longer sustain essential cell functions. On the other hand, loss of luminal Ca(2+) causes ER stress and activates an unfolded protein response, which, depending on the duration and severity of the stress, can reestablish normal ER function or lead to cell death. We will review these various diseases by mainly focusing on the mechanisms that cause ER Ca(2+) depletion.

Stone composition and metabolic status.

This paper aims to study the correlation between biochemical risk factors of the stone former and the type of oxalate stone formed, namely calcium oxalate monohydrate (COM) and calcium oxalate dehydrate (COD). A retrospective study of 487 patients who had been attending the urinary stone clinic, Trivandrum during 1998-2007 was conducted. The stones retrieved from them were subjected to chemical analysis and FTIR spectrographic analysis. They were categorized into COM, COD, mixed COM+COD and others. Of 142 pure calcium oxalate stone patients, 87 were predominantly COM stone formers and 55 COD stone formers. Their metabolic status of 24 h urine and serum was assessed. The values of urine calcium, phosphorus, uric acid, magnesium, creatinine, oxalate, citric acid, sodium and potassium, serum values of calcium, phosphorus, uric acid, magnesium and creatinine and calculated values of creatinine clearance, tubular reabsorption of phosphate, calcium magnesium ratio and calcium oxalate ratio were recorded. Comparison was made between the COM stone group and the COD stone group. Patients forming COM stones had significantly higher mean values for urine calcium (P < 0.05), oxalate (P < 0.01) and magnesium (P < 0.05) levels and significantly lower level of urine calcium-oxalate ratio (P < 0.01) and urine calcium-magnesium ratio (P < 0.01) compared to COD stone forming patients. All other values failed to show significant difference. Patients, with higher urine oxalate, formed COM stones. Those with low magnesium (which is an inhibitor) formed more of COD stones. Urine calcium was high in both groups without showing significant variation from the mean. In patients with high calcium-oxalate and calcium-magnesium ratios, there is higher chance of forming a COD stone than COM. Identification of the crystallization pattern of the calcium stone will help in selecting treatment modalities.

Case histories.

The conditions related to abnormalities of calcium and bone metabolism are large in number and cover problems of hypocalcaemia, hypercalcaemia, primary and secondary osteoporosis, rickets resulting from both vitamin D and phosphate metabolic disorders, and a series of miscellaneous conditions. Included in this chapter is a series of 36 cases drawn from our clinics and from colleagues who have presented these clinical problems at recent Advanced Courses in Paediatric Bone and Calcium Metabolism run by the British Paediatric and Adolescent Bone group. The series of cases is not fully comprehensive but is designed to cover the major aspects of bone and calcium related disorders and can be added to online in due course.

Calcium and vitamin D intakes may be positively associated with brain lesions in depressed and nondepressed elders.

Studies indicate that diet and vascular calcification may be related to the occurrence of brain lesions, although the importance of dietary calcium and vitamin D has not been investigated. The objective of this study was to test the hypothesis that calcium and vitamin D intakes would be positively associated with brain lesion volumes in elderly individuals with and without late-life depression. A cross-sectional study was performed as part of a longitudinal clinical study of late-life depression. Calcium and vitamin D intakes were assessed in 232 elderly subjects (95 with current or prior depression, 137 without depression) using a Block 1998 food frequency questionnaire. Calcium, vitamin D, and kilojoule intake were determined. Brain lesion volumes were calculated from magnetic resonance imaging scan. Subjects were 60 years or older. Calcium and vitamin D intakes were significantly and positively correlated with brain lesion volume (P < .05 and P < .001, respectively). In 2 separate multivariable models, controlling for age, hypertension, diabetes, heart disease, group (depression/comparison), lesion load (high/low), and total kilocalories, these positive associations remained significant (P < .05 for calcium; P < .001 for vitamin D). In conclusion, calcium and vitamin D consumption were associated with brain lesions in elderly subjects even after controlling for potentially explanatory variables. These associations may be due to vascular calcification or other mechanism. The possibility of adverse effects of high intakes of calcium and vitamin D needs to be further explored in longitudinal studies of elderly subjects.

Enhanced passive Ca2+ reabsorption and reduced Mg2+ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia.

Thiazide diuretics enhance renal Na+ excretion by blocking the Na+-Cl- cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca2+ transport in the proximal tubule rather than active Ca2+ transport in distal convolution explains thiazide-induced hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca2+ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced hypocalciuria in transient receptor potential channel subfamily V, member 5-knockout (Trpv5-knockout) mice, in which active distal Ca2+ reabsorption is abolished due to inactivation of the epithelial Ca2+ channel Trpv5. Third, HCTZ upregulated the Na+/H+ exchanger, responsible for the majority of Na+ and, consequently, Ca2+ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca2+ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of hypocalciuria parallels a compensatory increase in Na+ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg2+ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation.

Examination of megalin in renal tubular epithelium from patients with Dent disease.

Dent disease is characteristic for the urinary loss of low-molecular-weight proteins and calcium, leading to renal calcification and, in some patients, chronic renal failure. This disorder is caused by loss-of-function mutations in the renal chloride channel gene, CLCN5. The animal model of this disease has demonstrated the possible role of disturbed megalin expression, which is a member of the low-density lipoprotein receptor family and is associated with renal reabsorption of a variety of proteins, in Dent disease. We examined the expression of megalin in the renal tubular epithelium of two unrelated patients with Dent disease. One patient, whose CLCN5 gene was completely deleted, showed significantly decreased staining of megalin compared with controls, while there was no change in another patient with partial deletion of the gene. These results demonstrated that mutation of CLCN5 in some patients with Dent disease may impair the expression of megalin, resulting in abnormal calcium metabolism, manifested as hypercalciuria and nephrocalcinosis.

The calcium-sensing receptor in human disease.

The discovery of the calcium-sensing receptor (CaR), a G protein-coupled receptor, has led to the elucidation of the pivotal roles of the CaR in systemic calcium homeostasis. The receptor is situated on the chief cells of the parathyroid glands, where it senses the extracellular Ca2+ concentration and in turn alters the rate of secretion of parathyroid hormone (PTH). The intracellular signal pathways to which the CaR couples include, but are not limited to, phospholipase C (PLC), and mitogen-activated protein kinases. The receptor is widely expressed in various tissues and likely serves important cellular functions beyond that of maintaining systemic calcium homeostasis. Functionally important mutations in the receptor have been found to cause disorders in calcium homeostasis due both to changes in the set point for PTH secretion and to the control of renal calcium excretion. These mutations cause hypercalcemia when the mutation inactivates the receptor and cause hypocalcemia when the mutation activates the receptor. Recent studies have revealed the presence of circulating autoantibodies to the calcium-sensing receptor in humans, with the clinical presentation the same as that for diseases caused by mutations in the CaR. In renal secondary hyperparathyroidism, a drug that stimulates the receptor (calcimimetic) shows great promise as a medical treatment for this condition.

Disruption of the caveolin-1 gene impairs renal calcium reabsorption and leads to hypercalciuria and urolithiasis.

Using LoxP/Cre technology, we generated a knockout mouse homozygous for a null mutation in exon 2 of Cav1. In male Cav1-/- animals, we observed a dramatic increase in the incidence of urinary calcium stone formation. In 5-month-old male mice, the incidence of early urinary calculi was 67% in Cav1-/- mice compared to 19% in Cav1+/+ animals. Frank stone formation was observed in 13% of Cav1-/- males but was not seen in Cav1+/+ mice. Urine calcium concentration was significantly higher in Cav1-/- male mice compared to Cav1+/+ mice. In Cav1-/- mice, distal convoluted tubule cells were completely devoid of Cav1 and the localization of plasma membrane calcium ATPase was disrupted. Functional studies confirmed that active calcium absorption was significantly reduced in Cav1-/- compared to Cav1+/+ male mice. These results demonstrate that disruption of the Cav1 gene promotes the progressive steps required for urinary calcium stone formation and establish a new mouse model for urinary stone disease.

Feline endocrinology update.

This article highlights the advances in feline endocrinology, excluding diabetes mellitus and hyperadrenocorticism, which have recently been reviewed elsewhere. The goal will be to provide clinically relevant information regarding pathogenesis, diagnosis, and treatment options for these feline endocrine disorders.