Urinary proteomics for the study of genetic kidney diseases.

Despite their low prevalence, genetic kidney diseases (GKD) still represent a serious health problem. They often lead to kidney failure and to the consequent need of dialysis or kidney transplant. To date, reliable diagnosis requires laborious genetic tests and/or a renal biopsy. Moreover, only scant and non-specific markers exist for prognostic purposes. Biomarkers assayed in an easily available and low-cost sample, such as urine, would be highly valuable. Urinary proteomics can provide clues related to their development through the identification of differentially expressed proteins codified by the affected genes, or other dis-regulated species, in total or fractionated urine, providing novel mechanistic insights. In this review, the authors summarize and discuss the results of the main proteomic investigations on GKD urine samples and in urinary extracellular vesicles.

Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases.

Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications.

Quebec neonatal mass urinary screening programme: from micromolecules to macromolecules.

The Quebec Mass Urinary Screening Programme, initiated in 1971, has resulted in the screening of more than 2,500,000 newborns in the province of Quebec for 25 inherited Mendelian disorders divided into two groups. The first group concerns urea cycle disorders (citrullinaemia, hyperargininaemia, argininosuccinic aciduria), ketotic hyperglycinaemia, and organic acidurias (methylmalonic aciduria, glutaric aciduria type I, etc.); the second group relates to disorders of amino acid metabolism (cystathioninuria, prolidase deficiency, etc.) and transport (Fanconi syndrome, cystinurias, Hartnup syndrome, etc.). The main goal of the Programme is to detect and prevent these genetic diseases, some detectable only in urine, before the onset of clinical symptoms. A multiplex thin-layer chromatography methodology was developed, in which metabolites in urine are resolved and visualized by the sequential application of four different reagents to detect aminoacidopathies and organic acidurias. The technique is simple, reproducible, inexpensive and rapid, allowing the analysis of 500 samples daily by a single technician. The voluntary compliance of the parents is excellent, averaging 90% per year. Over the years, we have established a dynamic process, developing techniques or new reagents to detect as many treatable disorders as possible, now evaluating macromolecules associated with lysosomal storage disorders, mainly globotriaosylceramide (Gb3) for Fabry disease. We present here the methodology, infrastructure in place, results and recent statistics of the well-established Quebec Mass Urinary Screening Programme. We also report a study by tandem mass spectrometric analysis of urinary Gb3 in Fabry disease for the follow-up and monitoring of Fabry patients, as well as for its possible application to mass and high-risk screening programmes.

Diseases associated with calcium-sensing receptor.

The calcium-sensing receptor (CaSR) plays a pivotal role in systemic calcium metabolism by regulating parathyroid hormone secretion and urinary calcium excretion. The diseases caused by an abnormality of the CaSR are genetically determined or are more rarely acquired. The genetic diseases consist of hyper- or hypocalcemia disorders. Hypercalcaemia disorders are related to inactivating mutations of the CASR gene either heterozygous (autosomal dominant familial benign hypercalcaemia, still named hypocalciuric hypercalcaemia syndrome type 1) or homozygous (severe neonatal hyperparathyroidism). The A986S, R990G and Q1011E variants of the CASR gene are associated with higher serum calcium levels than in the general population, hypercalciuria being also associated with the R990G variant. The differential diagnosis consists in the hypocalciuric hypercalcaemia syndrome, types 2 (involving GNA11 gene) and 3 (involving AP2S1 gene); hyperparathyroidism; abnormalities of vitamin D metabolism, involving CYP24A1 and SLC34A1 genes; and reduced GFR. Hypocalcemia disorders, which are more rare, are related to heterozygous activating mutations of the CASR gene (type 1), consisting of autosomal dominant hypocalcemia disorders, sometimes with a presentation of pseudo-Bartter's syndrome. The differential diagnosis consists of the hypercalciuric hypocalcaemia syndrome type 2, involving GNA11 gene and other hypoparathyroidism aetiologies. The acquired diseases are related to the presence of anti-CaSR antibodies, which can cause hyper- or especially hypocalcemia disorders (for instance in APECED syndromes), determined by their functionality. Finally, the role of CaSR in digestive, respiratory, cardiovascular and neoplastic diseases is gradually coming to light, providing new therapeutic possibilities. Two types of CaSR modulators are known: CaSR agonists (or activators, still named calcimimetics) and calcilytic antagonists (or inhibitors of the CasR). CaSR agonists, such as cinacalcet, are indicated in secondary and primary hyperparathyroidism. Calcilytics have no efficacy in osteoporosis, but could be useful in the treatment of hypercalciuric hypocalcaemia syndromes.

Pathogenic uromodulin mutations result in premature intracellular polymerization.

Several renal diseases involve mutations in the gene encoding uromodulin, the predominant protein in urine. We investigated the intracellular processing of wild-type uromodulin, and three mutants: p.V93_G97del/ins AASC; C155R; and C150S. A renal biopsy from a patient harboring the C155R mutation revealed intracellular protein accumulation. Wild-type uromodulin was efficiently trafficked to the cell surface in transfected tsA 201 cells, whereas the mutants were partially retained within the cell, and incompletely processed. Atomic force microscopy imaging revealed that the intracellular mutant proteins contained fibrillar structures similar to urinary uromodulin. We suggest that premature intracellular polymerization underlies the pathology of uromodulin diseases.

A novel mutation in the flavin-containing monooxygenase 3 gene, FM03, that causes fish-odour syndrome: activity of the mutant enzyme assessed by proton NMR spectroscopy.

We have previously shown that primary trimethylaminuria, or fish-odour syndrome, is caused by an inherited defect in the flavin-containing monooxygenase 3 (FMO3) catalysed N-oxidation of the dietary-derived malodorous amine, trimethylamine (TMA). We now report a novel causative mutation for the disorder identified in a young girl diagnosed by proton nuclear magnetic resonance (NMR) spectroscopy of her urine. Sequence analysis of genomic DNA amplified from the patient revealed that she was homozygous for a T to C missense mutation in exon 3 of the FMO3 gene. The mutation changes an ATG triplet, encoding methionine, at codon 82 to an ACG triplet, encoding threonine. A polymerase chain reaction/restriction enzyme-based assay was devised to genotype individuals for the FMO3Thr82 allele. Wild-type and mutant FMO3, heterologously expressed in a baculovirus-insect cell system, were assayed by ultraviolet spectrophotometry and NMR spectroscopy for their ability to catalyse the N-oxidation of TMA. The latter technique has the advantage of enabling the simultaneous, direct and semi-continuous measurement of both of the products, TMA N-oxide and NADP, and of one of the reactants, NADPH. Results obtained from both techniques demonstrate that the Met82Thr mutation abolishes the catalytic activity of the enzyme and thus represents the genetic basis of the disorder in this individual. The combination of NMR spectroscopy with gene sequence and expression technology provides a powerful means of determining genotype-phenotype relationships in trimethylaminuria.

[Diagnosis of "fish odor syndrome" by urine nuclear magnetic resonance proton spectrometry]. / Diagnostic du "fish odour syndrome" par spectrométrie RMN proton des urines.

BACKGROUND: Trimethylaminuia is an unusual observation, often termed fish odor syndrome. The condition results from reduced ability to oxidize trimethylamine (TMA), which has a fishy odor, into odorless trimethylamine N-oxide (TMAO). METHOD: Proton nuclear magnetic resonance spectroscopy (MRS) was used as a simple and rapid method to detect TMA and TMAO in the same urine sample without pretreatment. Subjects were considered to have deficient N-oxidation of TMA if the TMAO/TMA ratio was greater than 80 p. 100 (heterozygous) or 65 p. 100 (homozygous). DISCUSSION: Direct proton RRS analysis of urine is well suited for diagnosis of fish odor syndrome. It can be used to detect heterozygous patients and also provides an easily implemented follow-up tool.

[Deficiency in medium chain acyl coA dehydrogenase manifested as febrile coma]. / Déficit en acyl-CoA déshydrogénase à chaîne moyenne révélé par un coma fébrile.

A 21-month-old infant developed coma with hypotonia during a viral infection. Acyl CoA dehydrogenase deficiency was diagnosed on the basis of results of the chromatographic study of organic acids performed on a urine specimen collected during the acute episode. However, other disorders of mitochondrial and fatty acid oxygenation can generate similar symptoms. Emphasis is put on the need for collecting urine specimens in patients who develop alterations in consciousness and hypoglycemia without ketonuria during prolonged fasting or repeated vomiting due to a viral infection. Urine chromatography can suggest which enzyme is defective, although the diagnosis should always be confirmed by a study of fatty acid oxygenation in lymphocytes or fibroblasts.

[Development of pregnancy in genetic risk patients with special reference to transabdominal amniocentesis]. / Die Entwicklung der Schwangerschaft bei genetischen Risikopatientinnen unter besonderer Berücksichtigung der transabdominalen Amniozentese.

A complex 3-stage-method of pregnancy evaluation in genetic patients with a planned-transabdominal amniocentesis reduces essentially the risk of the intervention; the state of maternal organism, placental function and fetal growth are repeatedly assessed. Fetal growth retardation together with poor placental function indicated in 4 cases a malformation of the type not be able to be diagnosed cytogeneticylla. Our experience is derived from 404 pregnancies followed before and after transabdominal amniocentesis.

Three cases of congenital adrenal hypoplasia: a cause of salt-wasting and mortality in the neonatal period.

Three infants with congenital adrenal hypoplasia are described. The two surviving infants were detected and successfully treated in the neonatal period due to a suggestive family history (Case 1) and antenatal maternal oestriol screening (Case 2). The modes of inheritance, diverse clinical presentation, associated conditions, diagnostic work-up and pathology of congenital adrenal hypoplasia in these three infants is discussed.

Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring.

BACKGROUND: N- and O-oligosaccharide variants on glycoproteins (glycoforms) can lead to alterations in protein activity or function that may manifest themselves as overt disease. APPROACH: This review summarizes those diseases that are known to be the result of an inherited or acquired glycoprotein oligosaccharide structural alteration and that are diagnosed in blood or urine by chemical characterization of that oligosaccharide alteration. CONTENT: The biochemical synthesis steps and catabolic pathways important in determining glycoprotein function are outlined with emphasis on alterations that lead to modified function. Clinical and biochemical aspects of the diagnosis are described for inherited diseases such as I-cell disease, congenital disorders of glycosylation, leukocyte adhesion deficiency type II, hereditary erythroblastic multinuclearity with a positive acidified serum test, and Wiskott-Aldrich syndrome. We also review the laboratory use of measurements of glycoforms related to acquired diseases such as alcoholism and cancer. CONCLUSIONS: Identification of glycoprotein glycoforms is becoming an increasingly important laboratory contribution to the diagnosis and management of human diseases as more diseases are found to result from glycan structural alterations.