Switch in FGFR3 and -4 expression profile during human renal development may account for transient hypercalcemia in patients with Sotos syndrome due to 5q35 microdeletions.

CONTEXT: Sotos syndrome is a rare genetic disorder with a distinct phenotypic spectrum including overgrowth and learning difficulties. Here we describe a new case of Sotos syndrome with a 5q35 microdeletion, affecting the fibroblast growth factor receptor 4 (FGFR4) gene, presenting with infantile hypercalcemia. OBJECTIVE: We strove to elucidate the evanescent nature of the observed hypercalcemia by studying the ontogenesis of FGFR3 and FGFR4, which are both associated with fibroblast growth factor (FGF) 23-mediated mineral homeostasis, in the developing human kidney. DESIGN: Quantitative RT-PCR and immunohistochemical analyses were used on archival human kidney samples to investigate the expression of the FGFR signaling pathway during renal development. RESULTS: We demonstrated that renal gene and protein expression of both FGFRs increased during fetal development between the gestational ages (GAs) of 14-40 weeks. Yet FGFR4 expression increased more rapidly as compared with FGFR3 (slope 0.047 vs 0.0075, P = .0018). Moreover, gene and protein expression of the essential FGFR coreceptor, Klotho, also increased with a significant positive correlation between FGFR and Klotho mRNA expression during renal development. Interestingly, we found that perinatal FGFR4 expression (GA 38-40 wk) was 7-fold higher as compared with FGFR3 (P = .0035), whereas in adult kidney tissues, FGFR4 gene expression level was more than 2-fold lower compared with FGFR3 (P = .0029), thus identifying a molecular developmental switch of FGFR isoforms. CONCLUSION: We propose that the heterozygous FGFR4 deletion, as observed in the Sotos syndrome patient, leads to a compromised FGF23 signaling during infancy accounting for transient hypercalcemia. These findings represent a novel and intriguing view on FGF23 mediated calcium homeostasis.

Pre-treatment of dairy and breast milk with sevelamer hydrochloride and sevelamer carbonate to reduce phosphate.

INTRODUCTION: Young children and infants with chronic kidney disease are at increased risk of hyperphosphatemia because of high intake of dairy products. Hyperphosphatemia leads to metastatic calcifications and an increased risk of cardiovascular complications. Sevelamer is an effective phosphate binder, but for children it has important practical disadvantages: it clogs enteral feeding tubes and can cause gastrointestinal complaints. Pre-treatment of dairy products to reduce their phosphate content might solve those problems. METHODS: Sevelamer hydrochloride and sevelamer carbonate were suspended in various dairy products (cow's milk, breast milk, baby formula, and tube-feeding formula). Each product was tested with varying concentrations of sevelamer. After suspension, each sample was stored for 10 minutes, allowing the sevelamer to precipitate. The supernatant was decanted and analyzed for pH and for phosphate, calcium, magnesium, potassium, sodium, and chloride content. RESULTS: We observed a significant decrease in the phosphate content of all tested products. With sevelamer hydrochloride, the phosphate reduction was 48% - 91% in the various products, and with sevelamer carbonate, it was 22% - 87%. The highest effectiveness was found in breast milk. A pH increase was found in all products. With sevelamer hydrochloride, a significant increase in chloride occurred. Notably, a significant decrease in calcium content (-75%) was observed in treated breast milk. CONCLUSIONS: Pretreatment of a variety of dairy products with either sevelamer hydrochloride or sevelamer carbonate effectively reduced their phosphate content and might avoid troublesome ingestion of sevelamer in children. The change in pH with sevelamer hydrochloride was remarkable, reflecting buffering mechanisms. The reduction in the calcium content of breast milk is a potential concern and should be carefully considered and monitored during clinical use of sevelamer.

Drug-induced alterations in Mg2+ homoeostasis.

Magnesium (Mg2+) balance is tightly regulated by the concerted actions of the intestine, bone and kidneys. This balance can be disturbed by a broad variety of drugs. Diuretics, modulators of the EGFR (epidermal growth factor receptor), proton pump inhibitors, antimicrobials, calcineurin inhibitors and cytostatics may all cause hypomagnesaemia, potentially leading to tetany, seizures and cardiac arrhythmias. Conversely, high doses of Mg2+ salts, frequently administered as an antacid or a laxative, may lead to hypermagnesaemia causing various cardiovascular and neuromuscular abnormalities. A better understanding of the molecular mechanisms underlying the adverse effects of these medications on Mg2+ balance will indicate ways of prevention and treatment of these adverse effects and could potentially provide more insight into Mg2+ homoeostasis.

The paradox of hyperdopaminuria in aromatic L-amino Acid deficiency explained.

Aromatic L-amino acid decarboxylase (AADC) decarboxylates 3,4-L-dihydroxylphenylalanine (L-dopa) to dopamine, and 5-hydroxytryptophan to serotonin. In AADC deficiency, dopamine and serotonin deficiency leads to a severe clinical picture with mental retardation, oculogyric crises, hypotonia, dystonia, and autonomic dysregulation. However, despite dopamine deficiency in the central nervous system, urinary dopamine excretion in AADC-deficient patients is normal to high.In human, renal AADC-activity is very high compared to other tissues including brain tissue. Plasma L-dopa levels are increased in AADC deficiency. In this study, the hypothesis that in AADC deficiency relatively high-residual renal AADC-activity combined with high substrate availability of L-dopa leads to normal or elevated levels of urinary dopamine is tested and verified using 24-h urine collection of two AADC-deficient patients.Renal dopamine is a major regulator of natriuresis and plays a crucial role in the maintenance of sodium homeostasis. Therefore, the preservation of sufficient renal AADC-activity in AADC deficiency might be crucial for survival of AADC-deficient patients.In this study, we underpinned an empirical finding with theory, thereby putting a clinical observation into its physiological context. Our study stresses the difference - not qualitatively but quantitatively - between dopamine production in the central nervous system and peripheral organs. Furthermore, this study clarifies the so far unexplained observation that neurotransmitter profiles in urine should be interpreted with extreme caution in the diagnostic work-up of patients suspected to suffer from neurometabolic disorders.

Apparent mineralocorticoid excess: time of manifestation and complications despite treatment.

Here we describe the case of a patient followed from birth because of a positive family history for apparent mineralocorticoid excess (AME) in an older brother. The patient, a girl, had normal serum electrolyte and blood pressure measurements in the first months after birth. Not until the age of 11 months did she develop anorexia and failure to thrive in combination with hypertension, hypokalemia, and metabolic alkalosis, which are consistent with the diagnosis of AME. This diagnosis was confirmed by mutation analysis of the HSD11B2 gene (C1228T). Treatment with amiloride and furosemide electrolyte disturbances normalized her blood pressure. At the age of 19 years she unexpectedly suffered a stroke. Additional investigations revealed no accepted risk factor for stroke. We discuss the possible underlying mechanisms for the delayed manifestation of hypertension and electrolyte disturbances in AME, propose an additional explanation for the stroke in this patient, and advise treatment with a mineralocorticoid receptor antagonist to reduce stroke risk in patients with AME.

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells.

Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.

Nephrogenic syndrome of inappropriate antidiuresis.

The nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare, recently recognized disorder in water balance affecting not only infants but also adults. A new molecular mechanism responsible for NSIAD has recently been identified: a gain of function of the arginine vasopressin (AVP) receptor type 2 (V2R), causing the constitutive activation of the receptor. Clinical presentation and laboratory findings of NSIAD resemble those of the syndrome of inappropriate secretion of antidiuretic hormone and consist of hyponatraemia, seizures and the lack of urinary dilution; however, the AVP levels in plasma are undetectable or very low. An elucidation of the pathophysiology of this syndrome will provide more insight into the action of AVP. An effective treatment of NSIAD is available. It consists of fluid restriction and administration of oral urea. Reported experience with furosemide treatment in NSIAD is still lacking.

Involvement of claudin 3 and claudin 4 in idiopathic infantile hypercalcaemia: a novel hypothesis?

BACKGROUND: Idiopathic infantile hypercalcaemia (IIH) is a rare disease that generally resolves spontaneously between the age of 1 and 3 years. Similar symptoms may occur in patients suffering from Williams-Beuren syndrome (WBS), which is caused by a microdeletion on chromosome 7. Two of the genes, named CLDN3 and CLDN4, located within this region are members of the claudin family that has been shown to be involved in paracellular calcium (Ca(2+)) absorption. Based on the hemizygous loss of CLDN3 and CLDN4 in WBS and the function of these genes in paracellular Ca(2+) transport, we hypothesized that mutations in CLDN3 or CLDN4 could also be involved in IIH. METHODS: Biochemical characteristics, including calciotropic hormone levels, were obtained from three typical IIH patients. CLDN3 and CLDN4 sequences were also analysed in these patients. The major intestinal Ca(2+) transporter TRPV6 was also screened for the presence of mutations, since hypercalcaemia in IIH and WBS has been shown to result from intestinal hyperabsorption. All three patients were also analysed for the presence of deletions or duplications using a single-nucleotide polymorphism (SNP) array for genomic DNA. RESULTS: The serum Ca(2+) levels of patients were 2.9, 3.3 and 3.8 mmol/L (normal <2.7 mmol/L). Levels of 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) were normal, parathyroid hormone (PTH) and PTH-related peptide (PTHrP) levels were appropriately low. Sequencing of coding regions and intron-exon boundaries did not reveal mutations in CLDN3, CLDN4 and TRPV6. Identified SNPs were not correlated with the disease phenotype. A SNP array did not reveal genomic deletions or duplications. CONCLUSIONS: Biochemical analysis did not reveal inappropriate levels of calciotropic hormones in IIH patients in this study. Furthermore, based on the lack of mutations in CLDN3, CLDN4 and TRPV6, we conclude that IIH is neither caused by mutations in these candidate genes nor by deletions or duplications in the genome of these patients.

Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters.

Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology.

Mitochondrial complex V expression and activity in cystinotic fibroblasts.

Alterations in ATP metabolism have been proposed to be involved in the pathogenesis of cystinosis, the most common form of inherited Fanconi syndrome. A recent study showed normal activity of respiratory chain complexes I-IV with decreased ATP levels in cystinotic fibroblasts. Here, we show normal complex V expression and activity in mitochondria of cystinotic fibroblasts. This indicates that alterations in mitochondrial oxidative phosphorylation enzymes are not responsible for ATP decrease in cystinotic fibroblasts.

Urinary protein excretion pattern and renal expression of megalin and cubilin in nephropathic cystinosis.

BACKGROUND: Nephropathic cystinosis is the most common cause of inherited renal Fanconi syndrome, caused by mutations in lysosomal cystine carrier cystinosin that result in lysosomal cystine accumulation throughout the body. How defects in cystinosin cause proximal tubular dysfunction is not known. We hypothesized that cystine accumulation could cause disturbed proximal tubular endocytosis by megalin and cubilin. STUDY DESIGN: Megalin, cubilin, and their ligands were studied in kidney tissue by means of immunohistochemistry. Urinary protein excretion pattern was evaluated. SETTING & PARTICIPANTS: Kidney tissue from a patient with cystinosis was compared with minimal change nephrotic syndrome tissue, end-stage renal disease tissue, and control renal tissue. Urine from 7 patients with cystinosis was compared with 6 control samples. RESULTS: Expression of megalin, cubilin, and ligands (transferrin, albumin, vitamin D-binding protein, alpha(1)-microglobulin, retinol-binding protein, and beta(2)-microglobulin) in convoluted proximal tubules of cystinotic kidney was similar to that in other kidney specimens. In straight tubules, low-molecular-weight proteins were present in only cystinotic kidney samples. Next to low-molecular-weight proteins and albumin, urinary excretion of immunoglobulin G was increased in patients with cystinosis with Fanconi syndrome compared with controls. This was already observed at an early age, suggesting enhanced glomerular permeability in patients with cystinosis. LIMITATIONS: This study is essentially observational, and immunohistochemical data are based on 1 cystinotic kidney. CONCLUSION: Our findings indicate that low-molecular-weight proteinuria in patients with cystinosis is not caused by decreased megalin and cubilin expression, and glomerular damage might already be present at early stages of the disease.

Proteomic profiling and identification in peritoneal fluid of children treated by peritoneal dialysis.

BACKGROUND: Proteomic technologies offer a high-throughput analysis of the expression of proteins in biological samples. The global analysis of the proteins in peritoneal dialysis (PD) fluid will provide a better understanding of the biological processes of the peritoneal membrane. METHODS: The dialysate of nine paediatric PD patients was collected from peritoneal equilibrium tests with 3.86% glucose. Proteins were separated on a 10% SDS-PAGE gel and in-gel digested with trypsin. Peptide mixtures were analysed using nanoLC-MS/MS and results were searched against the NCBI database. RESULTS: A total number of 189 proteins were identified in the PD fluid of nine patients, with 88 proteins shared by all patients. These 88 proteins accounted for 47% of the identified proteins and >90% of the total protein content in the analysed samples. Proteins were subdivided into eight different classes according to function. CONCLUSIONS: This study gives a representative overview of the proteins present in PD fluid. The proteins in PD fluid reflect plasma proteins as well as local peritoneal processes. Potentially interesting proteins are revealed.

Impaired routing of wild type FXYD2 after oligomerisation with FXYD2-G41R might explain the dominant nature of renal hypomagnesemia.

Autosomal dominant renal hypomagnesemia, associated with hypocalciurea, has been linked to a G to A mutation at nucleotide position 121 in the FXYD2 gene, resulting in the substitution of Gly with Arg at residue 41 of the protein. FXYD2, also called the Na,K-ATPase gamma-subunit, binds to Na,K-ATPase and influences its cation affinities. In this paper, we provide evidence for the molecular mechanism underlying the dominant character of the disorder. Co-immunoprecipitation experiments using tagged FXYD2 proteins demonstrated that wild type FXYD2 proteins oligomerise. Moreover, FXYD2-G41R also shows oligomerisation with itself and with the wild type protein. In the case of FXYD2-G41R, however, formation of homo-oligomers was prevented by addition of DTT or introduction of the C52A mutation. Finally, we demonstrated that artificial glycosylation of the wild type FXYD2 is reduced when co-expressed with FXYD2-G41R. These data indicate that binding of FXYD2-G41R to wild type FXYD2 subunit might abrogate the routing of wild type FXYD2 to the plasma membrane thus causing the dominant nature of this mutation.

Cystine dimethylester model of cystinosis: still reliable?

The ability of cystine dimethylester (CDME) to load lysosomes with cystine has been used to establish the basic defect in cystinosis: defective cystine exodus from lysosomes. Using CDME loading, it has been postulated that cystine accumulation in cystinosis affects mitochondrial ATP production, resulting in defective renal tubular reabsorption. Recent studies in cystinotic fibroblasts, however, show normal adenosine triphosphate (ATP) generation capacity. To investigate the effect of CDME in more detail, mitochondrial ATP generation, reactive oxygen species production, and viability are compared in fibroblasts loaded with CDME with those of cystinotic cells with a defective cystine transporter. Intracellular cystine levels were comparable in fibroblasts loaded with CDME (1 mM, 30 min) and cystinotic fibroblasts. Intracellular ATP levels and mitochondrial ATP production were decreased in fibroblasts loaded with CDME, but normal in cystinotic fibroblasts. Superoxide production was increased with 300% after CDME loading, whereas no changes were observed in cystinotic fibroblasts. Exposure to CDME led to cell death in a time- and concentration-dependent manner. Our data demonstrate that CDME has a toxic effect on mitochondrial ATP production and cell viability. These effects are not observed in cystinotic cells, indicating that a more appropriate model is required for studying the pathogenesis of cystinosis.

Interactions of Shiga-like toxin with human peripheral blood monocytes.

The cytotoxic effect of Shiga-like toxin (Stx; produced by certain Escherichia coli strains) plays a central role in typical hemolytic uremic syndrome (HUS). It damages the renal endothelium by inhibiting the cellular protein synthesis. Also, the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect. In this work, monocytes were studied as a potential transporter for Stx to the renal endothelium. Coincubation of isolated human monocytes loaded with Stx and target cells (vero cells and human umbilical vascular endothelial cells) were performed. Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry. Furthermore, the effect of a temperature shift on loaded monocytes was investigated. Stx-loaded monocytes reduced the protein synthesis of target cells. After adding an antibody against Stx, incomplete recovery occurred. Also, adding only the supernatant of coincubation was followed by protein synthesis inhibition. Stx detached from its receptor on the monocyte after a change in temperature, and no release was detected without this temperature shift. Although the monocyte plays an important role in the pathogenesis of HUS, it has no role in the transfer of Stx.

Adult and paediatric patients with minimal change nephrotic syndrome show no major alterations in glomerular expression of sulphated heparan sulphate domains.

BACKGROUND: Minimal change nephrotic syndrome (MCNS) is the most frequent form of nephrotic syndrome in childhood. In the glomerular basement membrane (GBM) of adult patients with MCNS, a reduced expression of a specific heparan sulphate (HS) domain has been reported. In children with MCNS, urinary activity of the HS-degrading enzyme heparanase was increased. It is, therefore, possible that a decreased GBM HS expression is associated with the pathogenesis of proteinuria in patients with MCNS. METHODS: In this study, HS in glomeruli of five adult and six paediatric patients with MCNS were analysed by immunofluorescence staining using four different antibodies, each defining a specific sulphated HS domain. The pediatric patients were subdivided into three groups depending on the presence or absence of podocyte foot process effacement, the level of proteinuria and prednisone administration at the time of the biopsy. In addition, kidneys of rats with adriamycin nephropathy (ADRN), a model for MCNS, were included in the study. RESULTS: Expression of sulphated HS domains was not aberrant in adult or paediatric patients compared with control subjects. Children with and without proteinuria had the same HS content. In contrast, rats with ADRN showed a decreased glomerular expression of sulphated HS domains. CONCLUSIONS: These results suggest that in patients with MCNS proteinuria is not associated with major changes in glomerular expression of sulphated HS domains.

In vivo degradation of heparan sulfates in the glomerular basement membrane does not result in proteinuria.

Heparan sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases, such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria. Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control). Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed. Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas removal of neuraminic acid does.

Lack of specific binding of Shiga-like toxin (verocytotoxin) and non-specific interaction of Shiga-like toxin 2 antibody with human polymorphonuclear leucocytes.

BACKGROUND: After gastrointestinal infection with Shiga-like toxin (Stx) producing Escherichia coli, the toxin is transported from the intestine to the renal microvascular endothelium. This is the main target for Stx in humans. Previous studies indicated that polymorphonuclear leucocytes (PMN) could serve as carriers for Stx in the systemic circulation. As at a later stage we could not confirm these data, we performed new studies. METHODS: The binding of Stx1 to PMN was determined in vitro (isolated human PMN and whole blood) and in vivo (injection in mice). The specificity of binding of an antibody against Stx2 to PMN from patients with haemolytic uraemic syndrome (HUS) was determined. This was compared with binding to PMN from healthy controls, and patients after haemodialysis (HD) or on peritoneal dialysis (PD). Furthermore, PMN were incubated with Stx to study possible activation. RESULTS: No specific binding of Stx1 to PMN could be detected. After intravenous injection of the toxin in mice, it was not associated with PMN. The binding of an antibody against Stx2 to PMN was detected in both patients with HUS and patients after HD, but not in patients on PD. Stx was not able to activate PMN. CONCLUSIONS: PMN are not acting as transporter for Stx in the pathogenesis of HUS. The interaction of a Stx antibody with PMN from HUS patients is not specific as it can also be observed in patients after HD (possibly due to activation of the PMN). Therefore, binding of Stx antibody to PMN is not reliable as a diagnostic tool for HUS.