Trafficking and Membrane Organization of GPI-Anchored Proteins in Health and Diseases.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of lipid-anchored proteins attached to the membranes by a glycolipid anchor that is added, as posttranslation modification, in the endoplasmic reticulum. GPI-APs are expressed at the cell surface of eukaryotes where they play diverse vital functions. Like all plasma membrane proteins, GPI-APs must be correctly sorted along the different steps of the secretory pathway to their final destination. The presence of both a glycolipid anchor and a protein portion confers special trafficking features to GPI-APs. Here, we discuss the recent advances in the field of GPI-AP trafficking, focusing on the mechanisms regulating their biosynthetic pathway and plasma membrane organization. We also discuss how alterations of these mechanisms can result in different diseases. Finally, we will examine the strict relationship between the trafficking and function of GPI-APs in epithelial cells.

[Inherited GPI deficiencies:a new disease with intellectual disability and epilepsy].

Glycosylphosphatidylinositol (GPI) is a glycolipid, which anchors 150 or more types of proteins to the cell surface. There are at least 26 genes involved in the biosynthesis and transport of GPI-anchored proteins (GPI-APs). Many inherited GPI deficiencies (IGDs) have been recently found using whole-exome sequencing. Patients with IGD have only a partial deficiency because complete GPI deficiency causes embryonic death. The major symptoms of IGDs include intellectual disability, epilepsy, coarse facial features, and multiple organ anomalies. These symptoms vary in severity depending upon the degree of the defect and/or position in the pathway of the affected gene. We clarified a mechanism of hyperphosphatasia, which is characterized by elevated release of tissue-nonspecific alkaline phosphatase. Hyperphosphatasia is observed in some patients with IGDs, such as hyperphosphatasia mental retardation syndrome or Mabry syndrome, caused by mutations in genes in the later stage of GPI biosynthesis. The possibility of IGD should be considered in patients with seizures and intellectual disability. The presence of hyperphosphatasia is strong evidence of IGD. Flow cytometric analysis of GPI-APs on granulocytes is also useful for the detection of IGD.

Clinical assessment of phosphorus status, balance and renal handling in normal individuals and in patients with chronic kidney disease.

PURPOSE OF REVIEW: The concepts of steady state, external balance, total body status and internal distribution are not always appreciated or even considered in clinical practice. The current tests available for clinical assessment of phosphorus physiology and pathophysiology are valid in some aspects, but also have many limitations. The purpose of this review is to clarify the above concepts and discuss the utility of the currently available tests to assess phosphorus disorders. RECENT FINDINGS: Both epidemiologic and preclinical data have shown that disturbances in mineral metabolism contribute significantly to the morbidity and mortality in chronic kidney disease. There are also emerging data supporting the notion that phosphotoxicity may exist even in individuals with normal renal function. In chronic kidney disease (CKD), hyperphosphatemia is a relative late event and is a suboptimal indicator of phosphorus balance and status. The judicious use of plasma and urine chemistry and hormonal biomarkers such as fibroblast growth factor 23 should be considered. SUMMARY: There is a dire need to increase awareness of what physiologic parameters should be monitored in terms of phosphorus pathophysiology. Although the current available tests in the clinical armamentarium are not ideal, understanding their implications and limitations will improve patient care and motivate practitioners and investigators to develop better tests.

FGF-23: the rise of a novel cardiovascular risk marker in CKD.

Elevated plasma levels of the phosphaturic hormone fibroblast growth factor 23 (FGF-23) are a hallmark of chronic kidney disease (CKD)-mineral and bone disorder. FGF-23 allows serum phosphate levels within physiological limits to be maintained in progressive CKD until end-stage renal disease is reached. Despite its seemingly beneficial role in phosphate homeostasis, several prospective studies in dialysis patients and in patients with less advanced CKD associated elevated FGF-23 with poor cardiovascular and renal outcome. Moreover, very recent evidence suggests an adverse prognostic impact of elevated FGF-23 even in subjects without manifest CKD. These epidemiological data are supplemented by laboratory findings that reveal a pathophysiological role of FGF-23 in the pathogenesis of myocardial injury. In aggregate, these clinical and experimental data identify FGF-23 as a promising target of novel therapeutic interventions in CKD and beyond, which should be tested in future clinical trials.

Phosphorus metabolism disorders in erythrocytes and lymphocytes among patients with inflammatory breast cancer, infiltrative stomach and colorectal cancer.

Energy and plastic potential dysfunction of erythrocytes and lymphocytes among people with inflammatory breast cancer, infiltrative stomach cancer, and infiltrative colon cancer is characterized by a more aggressive clinical course and poor prognosis. We explored the features of energy metabolism and phosphorus metabolism disorders in the erythrocytes and lymphocytes of patients with inflammatory breast cancer, infiltrative stomach cancer, and infiltrative colon cancer as a predicting factor in the course of the disease. 49 people were examined; the 1st group had infiltrative stomach cancer (n=17); the 2nd group had infiltrative colon cancer (n=11); the 3rd group had inflammatory breast cancer (n=21). Glycerol-3-phosphate dehydrogenase activity was 1.8 times reduced (p≤0.005), and the activity of glyceraldehyde-3-phosphate dehydrogenase in erythrocytes of patients with cancer at the main localization increased 2.5 times, compared with normal. Inflammatory breast cancer patients had a statistically significant decrease (p<0.005) in erythrocytes adenosine triphosphate content by an average of 56.5% compared with the normal ratio, and in cases of patients with gastric and colorectal cancer, a decrease of 67%. Excessive use of phosphorus for energy metabolism and adenosine triphosphate production destroys the balance of energetic and plastic potentials of erythrocytes and lymphocytes in inflammatory breast cancer, infiltrative stomach, and infiltrative colorectal cancers patients.

Clinical practice. Fibroblast growth factor (FGF)23: a new hormone.

Until a decade ago, two main hormones were recognized as directly affecting phosphate homeostasis and, with that, bone metabolism: parathyroid hormone and 1,25(OH)(2) vitamin D (calcitriol). It was only a decade ago that the third major player hormone was found, linking gut, bone, and kidney. The physiologic role of fibrinogen growth factor (FGF)23 is to maintain serum phosphate concentration within a narrow range. Secreted from osteocytes, it modulates kidney handling of phosphate reabsorption and calcitriol production. Genetic and acquired abnormalities in FGF23 structure and metabolism cause conditions of either hyper-FGF23-manifested by hypophosphatemia, low serum calcitriol, and rickets/osteomalacia-or hypo-FGF23, expressed by hyperphosphatemia, high serum calcitriol, and extra-skeletal calcifications. In patients with chronic renal failure, FGF23 levels increase as kidney functions deteriorate and are under investigation to learn if the hormone actually participates in the pathophysiology of the deranged bone and mineral metabolism typical for these patients and, if so, whether it might serve as a therapeutic target. This review addresses the physiology and pathophysiology of FGF23 and its clinical applications.

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.

PGAP3 Associated with Hyperphosphatasia with Mental Retardation Plays a Novel Role in Brain Morphogenesis and Neuronal Wiring at Early Development.

Recessive mutations in Post-GPI attachment to proteins 3 (PGAP3) cause the rare neurological disorder hyperphosphatasia with mental retardation syndrome 4 type (HPMRS4). Here, we report a novel homozygous nonsense mutation in PGAP3 (c.265C>T-p.Gln89*), in a 3-year-old boy with unique novel clinical features. These include decreased intrauterine fetal movements, dysgenesis of the corpus callosum, olfactory bulb agenesis, dysmorphic features, cleft palate, left ear constriction, global developmental delay, and hypotonia. The zebrafish functional modeling of PGAP3 loss resulted in HPMRS4-like features, including structural brain abnormalities, dysmorphic cranial and facial features, hypotonia, and seizure-like behavior. Remarkably, morphants displayed defective neural tube formation during the early stages of nervous system development, affecting brain morphogenesis. The significant aberrant midbrain and hindbrain formation demonstrated by separation of the left and right tectal ventricles, defects in the cerebellar corpus, and caudal hindbrain formation disrupted oligodendrocytes expression leading to shorter motor neurons axons. Assessment of zebrafish neuromuscular responses revealed epileptic-like movements at early development, followed by seizure-like behavior, loss of touch response, and hypotonia, mimicking the clinical phenotype human patients. Altogether, we report a novel pathogenic PGAP3 variant associated with unique phenotypic hallmarks, which may be related to the gene's novel role in brain morphogenesis and neuronal wiring.

Phosphate binders in chronic kidney disease and end-stage renal disease: a patient-centered approach.

Disorders of calcium and phosphorus metabolism are associated with significant morbidity and mortality in patients with advanced chronic kidney disease. These patients typically require oral phosphate binders to maintain phosphorus homeostasis, but the choice of which among several agents to use has been actively investigated and debated. Recent debate has been polarized between those who favor calcium-based binders for their proven efficacy and relatively low cost and those who favor sevelamer for its putative beneficial effects on inflammatory biomarkers and vascular calcification. This review summarizes the current state of the art of prescribing phosphate binders, ranging from large-scale clinical trials to focused mechanistic studies, and proposes that the available evidence does not conclusively prove the relative superiority of any one binder.

Phosphate homeostasis disorders.

Our understanding of the regulation of phosphate balance has benefited tremendously from the molecular identification and characterization of genetic defects leading to a number of rare inherited or acquired disorders affecting phosphate homeostasis. The identification of the key phosphate-regulating hormone, fibroblast growth factor 23 (FGF23), as well as other molecules that control its production, such as the glycosyltransferase GALNT3, the endopeptidase PHEX, and the matrix protein DMP1, and molecules that function as downstream effectors of FGF23 such as the longevity factor Klotho and the phosphate transporters NPT2a and NPT2c, has permitted us to understand the complex interplay that exists between the kidneys, bone, parathyroid, and gut. Such insights from genetic disorders have allowed not only the design of potent targeted treatment of FGF23-dependent hypophosphatemic conditions, but also provide clinically relevant observations related to the dysregulation of mineral ion homeostasis in health and disease.

Characterization of glycosylphosphatidylinositol biosynthesis defects by clinical features, flow cytometry, and automated image analysis.

BACKGROUND: Glycosylphosphatidylinositol biosynthesis defects (GPIBDs) cause a group of phenotypically overlapping recessive syndromes with intellectual disability, for which pathogenic mutations have been described in 16 genes of the corresponding molecular pathway. An elevated serum activity of alkaline phosphatase (AP), a GPI-linked enzyme, has been used to assign GPIBDs to the phenotypic series of hyperphosphatasia with mental retardation syndrome (HPMRS) and to distinguish them from another subset of GPIBDs, termed multiple congenital anomalies hypotonia seizures syndrome (MCAHS). However, the increasing number of individuals with a GPIBD shows that hyperphosphatasia is a variable feature that is not ideal for a clinical classification. METHODS: We studied the discriminatory power of multiple GPI-linked substrates that were assessed by flow cytometry in blood cells and fibroblasts of 39 and 14 individuals with a GPIBD, respectively. On the phenotypic level, we evaluated the frequency of occurrence of clinical symptoms and analyzed the performance of computer-assisted image analysis of the facial gestalt in 91 individuals. RESULTS: We found that certain malformations such as Morbus Hirschsprung and diaphragmatic defects are more likely to be associated with particular gene defects (PIGV, PGAP3, PIGN). However, especially at the severe end of the clinical spectrum of HPMRS, there is a high phenotypic overlap with MCAHS. Elevation of AP has also been documented in some of the individuals with MCAHS, namely those with PIGA mutations. Although the impairment of GPI-linked substrates is supposed to play the key role in the pathophysiology of GPIBDs, we could not observe gene-specific profiles for flow cytometric markers or a correlation between their cell surface levels and the severity of the phenotype. In contrast, it was facial recognition software that achieved the highest accuracy in predicting the disease-causing gene in a GPIBD. CONCLUSIONS: Due to the overlapping clinical spectrum of both HPMRS and MCAHS in the majority of affected individuals, the elevation of AP and the reduced surface levels of GPI-linked markers in both groups, a common classification as GPIBDs is recommended. The effectiveness of computer-assisted gestalt analysis for the correct gene inference in a GPIBD and probably beyond is remarkable and illustrates how the information contained in human faces is pivotal in the delineation of genetic entities.

Pyrophosphohydrolase activity and inorganic pyrophosphate content of cultured human skin fibroblasts. Elevated levels in some patients with calcium pyrophosphate dihydrate deposition disease.

In calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, metabolic abnormalities favoring extracellular inorganic pyrophosphate (PPi) accumulation have been suspected. Elevations of intracellular PPi in cultured skin fibroblasts from a single French kindred with familial CPPD deposition (19) and elevated nucleoside triphosphate pyrophosphohydrolase activity (NTPPPH), which generates PPi in extracts of CPPD crystal-containing cartilages (14) favor this suspicion. To determine whether NTPPPH activity or PPi content of cells might be a disease marker expressed in extraarticular cells, human skin-derived fibroblasts were obtained from control donors and patients affected with the sporadic and familial varieties of CPPD (CPPD-S and CPPD-F) deposition. Intracellular PPi was elevated in both CPPD-S (P less than 0.05) and CPPD-F (P less than 0.01) fibroblasts compared with control fibroblasts. Ecto-NTPPPH activity was elevated in CPPD-S (P less than 0.01) but not CPPD-F. Intracellular PPi correlated with ecto-NTPPPH (P less than 0.01). Elevated PPi levels in skin fibroblasts may serve as a biochemical marker for patients with familial or sporadic CPPD crystal deposition disease; ecto-NTPPPH activity further separates the sporadic and familial disease types. Expression of these biochemical abnormalities in nonarticular cells implies a generalized metabolic abnormality.

Phosphate salivary secretion in hemodialysis patients: implications for the treatment of hyperphosphatemia.

BACKGROUND/AIMS: Hyperphosphatemia is recognized as contributing to the increased risk of cardiac death in end-stage renal disease (ESRD) and hemodialysis (HD) patients. Currently available pharmacologic treatment for hyperphosphatemia is based on phosphate binders but, despite treatment, only half of the patients fall within the range for serum phosphorus of the K/DOQI guidelines. Therefore, there is a need to identify other therapeutic approaches in order to reduce serum phosphate. Salivary fluid contains phosphate which, if related to the daily salivary secretion (1,000-1,880 ml), may raise interest in order to identify further additive approaches to phosphorus removal in uremic patients, while data about salivary phosphate secretion in ESRD patients are controversial. METHODS: This study evaluates salivary phosphate secretion in 68 HD patients compared with 30 healthy subjects. Saxon's test confirmed normal salivary function in patients and controls. Salivary calcium and serum phosphate, calcium and PTH were also measured. RESULTS: HD patients had significantly higher salivary phosphorus levels compared with healthy controls: 30.35 (26.5-34.6) vs. 12.1 (10.58-14.73) mg/dl (p < 0.0001), and this significantly correlated (p < 0.0001) with serum phosphorus. Multiple regression analysis confirmed serum phosphorus as the only predictor (p < 0.0001) of salivary phosphorus. CONCLUSIONS: Given the functional secretive similarity between salivary glands and the kidneys, this increased salivary phosphate secretion might be interpreted as being compensatory in the presence of renal failure. Absorption of the increased salivary phosphate secretion, however, may worsen hyperphosphatemia; therefore, the binding of salivary phosphate might be considered as a further therapeutic approach to hyperphosphatemia in ESRD.

Hyperphosphatemia and phosphate binders.

PURPOSE: The pathophysiology of hyperphosphatemia associated with end-stage renal disease and treatment with phosphate binders are discussed. SUMMARY: Phosphorus is an essential element necessary for the normal function of the human body, required for skeletal construction and synthesis of DNA, proteins, and adenosine triphosphate. In healthy individuals, serum phosphorus concentrations are maintained between 2.5 and 4.5 mg/dL through diet and renal excretion. In renal insufficiency, phosphorus excretion declines and hyperphosphatemia develops. The body's compensation mechanisms cause secondary hyperparathyroidism and renal osteodystrophy. Phosphate binders provide an effective means for managing serum phosphate. Commercially available phosphate binders include calcium carbonate, calcium acetate, sevelamer, lanthanum, and, rarely, aluminum hydroxide. Because of aluminum's known toxicities, aluminum-based phosphate binders have a limited place in therapy. Calcium carbonate's benefits are seen over a narrow gastric pH range, thereby limiting the drug's utility. Calcium acetate is effective over a wide pH range. Other phosphate binders, including sevelamer hydrochloride and lanthanum carbonate, have recently entered the market, but their use remains controversial. CONCLUSION: If left untreated, hyperphosphatemia can result in secondary hyperparathyroidism, renal osteodystrophy, and metastatic calcification of blood vessels and soft tissue. The treatment of hyperphosphatemia in patients with chronic renal failure includes dialysis, dietary phosphorus restrictions, phosphate-binding medications, and vitamin D analogs. Selection of phosphate binders should be based on patient characteristics, including serum phosphate, serum calcium, and intact parathyroid hormone concentrations, and patient tolerability.

Phosphate diabetes, tubular phosphate reabsorption and phosphatonins.

Phosphate diabetes is defined as inadequate tubular reabsorption. Hypophosphatemia is responsible for most of the clinical manifestations, which vary with the age of the patient and the severity of the phosphate wasting. Vitamin D-resistant rickets in children or osteomalacia in adults, osteoporosis, bone pain including spinal pain, and pain in the joints and periarticular areas are the main manifestations. Several factors are known to affect tubular phosphate reabsorption via the sodium/phosphate cotransporters located on the tubular cell membranes. Factors that decrease phosphate reabsorption include a high intake of dietary phosphate, acidosis, parathyroid hormone (PTH), PTH-related peptide (PTHrp), glucocorticoid therapy, calcitonin, and vitamin D. On the other hand, a low-phosphate diet, alkalosis, growth hormone, insulin, IGF-1, and thyroid hormones increase tubular phosphate reabsorption. Physiological concepts about tubular phosphate reabsorption have been radically changed by the recent identification of phosphaturic factors called phosphatonins. The most extensively studied phosphatonin to date is fibroblast growth factor 23 (FGF23), which was first identified in patients with tumor-induced osteomalacia and shown to be secreted by the neoplastic cells. The FGF23 has also been implicated in autosomal dominant hypophosphatemic rickets, in which a gene mutation results in production of abnormal FGF23 that resists hydrolysis. In healthy individuals, FGF23 contributes to regulate phosphate reabsorption via Na/Pi cotransporters. Other phosphatonins may exist, such as matrix extracellular phosphoglycoprotein (MEPE) and secreted frizzled-related protein 4 (SFRP4), whose role remains to be defined. The part played by these proteins in idiopathic renal phosphate wasting in adults needs to be investigated.

[News about phosphorus metabolism]. / Nouveautés a propos du métabolisme du phosphore.

Phosphorus is mainly present in the serum as phosphate which is the principal intracellular anion, essential for the organism, in particular through its role in the production of ATP. In plasma, only inorganic phosphorus (Pi) is measured. In this article, we will review new information about the regulation of Pi at the intestinal, osseous and mainly renal levels. The various cotransporters sodium-phosphorus (Na-Pi) will also be examined and the molecular regulation mechanisms approached. Finally, illustration of Pi physiology will be given through various clinical examples.

Expansile skeletal hyperphosphatasia is caused by a 15-base pair tandem duplication in TNFRSF11A encoding RANK and is allelic to familial expansile osteolysis.

Expansile skeletal hyperphosphatasia (ESH) is a singular disorder characterized in the year 2000 in a mother and daughter with early-onset deafness, premature loss of teeth, progressive hyperostotic widening of long bones causing painful phalanges in the hands, accelerated bone remodeling, and episodic hypercalcemia likely inherited as a highly penetrant, autosomal dominant trait. Absence of large osteolytic lesions with cortical thinning in major long bones, together with bouts of hypercalcemia, indicated that ESH is not a variant of familial expansile osteolysis (FEO). Here, we investigated the molecular basis of ESH after three families with FEO were reported to have an identical 18-base pair tandem duplication (84dup18) in the signal peptide sequence of the TNFRSF11A gene that encodes receptor activator of nuclear factor-kappaB (RANK). We find that ESH is caused by a remarkably similar 15-base pair tandem duplication (84dup15) in TNFRSF11A. Hence, ESH and FEO are allelic diseases and ESH, like FEO, probably reflects increased activity in the skeleton of the RANK target, nuclear factor-kappaB (NF-kappaB).

The mobilization of bone mineral by 1,25-dihydroxyvitamin D3 in hypophosphatemic rats.

Administration of 1,25-dihydroxyvitamin D3 to thyroparathyroidectomized, hypophosphatemic rats on a low-phosphorus diet increases serum inorganic phosphorus and calcium concentrations. Experiments utilizing such rats treated with 45Ca and 32P ten days before use revealed that the rise in serum phosphorus in response in 1,25-dihydroxy-vitamin D3 is derived at least in part from bone mineral. This suggests that under conditions of hypophosphatemia, 1,25-dihydroxyvitamin D3 can mobilize bone in the absence of parathyroid hormone.

Acquired hypophosphatemia.

This article discusses the regulation of serum phosphorus under normal conditions, focusing on the pathophysiology of acquired hypophosphatemia and its clinical manifestation. In addition, the clinical settings and conditions in which hypophosphatemia is likely to cause severe morbidity are emphasized.