Weibel-Palade bodies: function and role in thrombotic thrombocytopenic purpura and in diarrhea phase of STEC-hemolytic uremic syndrome.

Vascular endothelial cells are equipped with numerous specialized granules called Weibel-Palade bodies (WPBs). They contain a cocktail of proteins that can be rapidly secreted (3-5 min) into the vascular lumen after an appropriate stimulus such as thrombin. These proteins are ready without synthesis. Von Willebrand factor (VWF) and P-selectin are the main constituents of WPBs. Upon stimulation, release of ultralarge VWF multimers occurs and assembles into VWF strings on the apical side of endothelium. The VWF A1 domain becomes exposed in a shear-dependent manner recruiting and activating platelets. VWF is able to recruit leukocytes via direct leukocyte binding or via the activated platelets promoting NETosis. Ultralarge VWF strings are ultimately cleaved into smaller pieces by the protease ADAMTS-13 preventing excessive platelet adhesion. Under carefully performed flowing conditions and adequate dose of Shiga toxins, the toxin induces the release of ultralarge VWF multimers from cultured endothelial cells. This basic information allows insight into the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and of STEC-HUS in the diarrhea phase. In TTP, ADAMTS-13 activity is deficient and systemic aggregation of platelets will occur after a second trigger. In STEC-HUS, stimulated release of WPB components in the diarrhea phase of the disease can be presumed to be the first hit in the damage of Gb3 positive endothelial cells.

The cause of eyelid ptosis, orthostatic hypotension and exercise intolerance.

To provide more insight in the delay in diagnosis and expectation of treatment adapted for the paediatrician, the data were collected from patients described with dopamine beta-hydroxylase deficiency are evaluated. More insight in clinical features of dopamine beta-hydroxylase deficiency consisting mainly of eyelid ptosis, orthostatic hypotension, hypoglycaemia and exercise intolerance, explains the delay in diagnosis of this congenital disorder, although all symptoms some more concealed are present. An increasing experience by L-DOPS, a resurrection for the patient, allows recommendations for early treatment. An explanation for the delay in diagnosis is provided together with the advice for treatment.

Human and animal fertility studies in cystinosis reveal signs of obstructive azoospermia, an altered blood-testis barrier and a subtherapeutic effect of cysteamine in testis.

Cystinosis is an inherited metabolic disorder caused by autosomal recessive mutations in the CTNS gene leading to lysosomal cystine accumulation. The disease primarily affects the kidneys followed by extra-renal organ involvement later in life. Azoospermia is one of the unclarified complications which are not improved by cysteamine, which is the only available disease-modifying treatment. We aimed at unraveling the origin of azoospermia in cysteamine-treated cystinosis by confirming or excluding an obstructive factor, and investigating the effect of cysteamine on fertility in the Ctns-/- mouse model compared with wild type. Azoospermia was present in the vast majority of infantile type cystinosis patients. While spermatogenesis was intact, an enlarged caput epididymis and reduced levels of seminal markers for obstruction neutral α-glucosidase (NAG) and extracellular matrix protein 1 (ECM1) pointed towards an epididymal obstruction. Histopathological examination in human and mouse testis revealed a disturbed blood-testis barrier characterized by an altered zonula occludens-1 (ZO-1) protein expression. Animal studies ruled out a negative effect of cysteamine on fertility, but showed that cystine accumulation in the testis is irresponsive to regular cysteamine treatment. We conclude that the azoospermia in infantile cystinosis is due to an obstruction related to epididymal dysfunction, irrespective of the severity of an evolving primary hypogonadism. Regular cysteamine treatment does not affect fertility but has subtherapeutic effects on cystine accumulation in testis.

Blood, urine and cerebrospinal fluid analysis in TH and AADC deficiency and the effect of treatment.

BACKGROUND: Aromatic L-amino acid decarboxylase (AADC) deficiency and tyrosine hydroxylase (TH) deficiency are rare inherited disorders of monoamine neurotransmitter synthesis which are typically diagnosed using cerebrospinal fluid examination of monoamine neurotransmitter metabolites. Until now, it has not been systematically studied whether analysis of monamine neurotransmitter metabolites in blood or urine has diagnostic value as compared to cerebrospinal fluid examination, or whether monoamine neurotransmitter metabolites in these peripheral body fluids is useful to monitor treatment efficacy. METHODS: Assessment, both by literature review and retrospective analysis of our local university hospital database, of monoamine neurotransmitter metabolites in urine, blood and cerebrospinal fluid, and serum prolactin levels, before and during treatment in patients with AADC and TH deficiency. RESULTS: In AADC deficiency, 3-O-methyldopa in serum or dried blood spots was reported in 34 patients and found to be (strongly) increased in all, serotonin in serum was decreased in 7/7 patients. Serum prolactin was increased in 34/37 and normal in 3 untreated patients. In urine, dopamine was normal or increased in 21/24 patients, 5-hydroxyindoleacetic acid was decreased in 9/10 patients, and vanillactic acid was increased in 19/20 patients. No significant changes were seen in monoamine neurotransmitter metabolites after medical treatment, except for an increase of homovanillic acid in urine and cerebrospinal fluid after levodopa therapy, sometimes even in absence of a clinical response. After gene therapy, cerebrospinal fluid homovanillic acid increased in most patients (8/12), but 5-hydroxyindoleacetic acid remained unchanged in 9/12 patients.In TH deficiency, serum prolactin was increased in 12/14 and normal in the remaining untreated patients. Urinary dopamine was decreased in 2/8 patients and normal in 6. Homovanillic acid concentrations in cerebrospinal fluid increased upon levodopa treatment, even in the absence of a clear treatment response. CONCLUSIONS: This study confirms that cerebrospinal fluid is the most informative body fluid to measure monoamine neurotransmitter metabolites when AADC or TH deficiency is suspected, and that routine follow-up of cerebrospinal fluid measurements to estimate treatment response is not needed. 3-O-methyldopa in dried blood spots and vanillactic acid in urine are promising peripheral biomarkers for diagnosis of AADC deficiency. However, in many patients with TH or AADC deficiency dopamine in urine is normal or increased thereby not reflecting the metabolic block. The value of serum prolactin for follow-up of AADC and TH deficiency should be further studied.

Clinical presentation and long-term follow-up of dopamine beta hydroxylase deficiency.

Dopamine beta hydroxylase (DBH) deficiency is an extremely rare autosomal recessive disorder with severe orthostatic hypotension, that can be treated with L-threo-3,4-dihydroxyphenylserine (L-DOPS). We aimed to summarize clinical, biochemical, and genetic data of all world-wide reported patients with DBH-deficiency, and to present detailed new data on long-term follow-up of a relatively large Dutch cohort. We retrospectively describe 10 patients from a Dutch cohort and 15 additional patients from the literature. We identified 25 patients (15 females) from 20 families. Ten patients were diagnosed in the Netherlands. Duration of follow-up of Dutch patients ranged from 1 to 21 years (median 13 years). All patients had severe orthostatic hypotension. Severely decreased or absent (nor)epinephrine, and increased dopamine plasma concentrations were found in 24/25 patients. Impaired kidney function and anemia were present in all Dutch patients, hypomagnesaemia in 5 out of 10. Clinically, all patients responded very well to L-DOPS, with marked reduction of orthostatic complaints. However, orthostatic hypotension remained present, and kidney function, anemia, and hypomagnesaemia only partially improved. Plasma norepinephrine increased and became detectable, while epinephrine remained undetectable in most patients. We confirm the core clinical characteristics of DBH-deficiency and the pathognomonic profile of catecholamines in body fluids. Impaired renal function, anemia, and hypomagnesaemia can be part of the clinical presentation. The subjective response to L-DOPS treatment is excellent and sustained, although the neurotransmitter profile in plasma does not normalize completely. Furthermore, orthostatic hypotension as well as renal function, anemia, and hypomagnesaemia improve only partially.

Serum Shiga toxin 2 values in patients during acute phase of diarrhoea-associated haemolytic uraemic syndrome.

AIM: Shiga toxins are delivered via systemic circulation and are considered to be the cause of diarrhoea-associated haemolytic uraemic syndrome (HUS), as they injure endothelial cells, particularly in the glomeruli. This study measured Shiga toxin 2 (Stx2) in the serum of children affected in by HUS due to Stx2 producing Escherichia coli. METHODS: The concentration of free Stx2 was measured in the serum of 16 children, collected immediately after admission to the clinic in the acute phase of HUS, using a sandwich enzyme-linked immunosorbent assay. The family members of two children were also investigated, with the relative toxicity of Stx2 assessed by a Vero cell-based fluorescent assay. RESULTS: Stx2 was found in the serum of eight of the 16 children who were investigated. It was also detected in four of the six family members not showing symptomatic HUS, with an extremely high level in two. CONCLUSION: An absent or rather low concentration of Stx2 was found in the serum of children admitted to the clinic with diarrhoea-associated HUS. The high concentration of Stx2 in family members without HUS, but mostly with watery diarrhoea and raised functional activity, was in line with the concept of early injury by Stx2.

Urinary excretion of polyols and sugars in children with chronic kidney disease.

BACKGROUND: The urinary concentrations of monosaccharides and polyols are used for diagnosing inborn errors of metabolism and renal tubular disorders. Reference values are age-related and depend on the method of detection. However, the influence of the renal function is often still neglected. In this study we examined the urinary excretion of monosaccharides and polyols in children with various degrees of chronic kidney disease (CKD), but with no known metabolic or renal tubular disorders. CASE DIAGNOSIS/TREATMENT: In 25 patients with CKD stage 1-5, urinary concentrations of 18 monosaccharides and polyols were measured by gas chromatography-mass spectrometry (GC-MS) in random urinary samples and were compared with age-related reference values. Serum creatinine was measured at the time of the urine sample, and the height-independent estimated glomerular filtration rate (eGFR-Pottel) was calculated. Urinary excretions of monosaccharides and polyols were above the reference values in 8-88% of all patients. A significant difference between CKD stage 1-2 compared with CKD stage 3-5 was found for allose, arabitol and sorbitol (p < 0.05) and for arabinose, fucose, myoinositol, ribitol, xylitol, and xylose (p < 0.01). CONCLUSIONS: We show that the excretion of polyols and sugars depends on eGFR, which warrants a cautious interpretation of the results in patients with CKD.

Segmental transport of Ca²âº and Mg²âº along the gastrointestinal tract.

Calcium (Ca(2+)) and magnesium (Mg(2+)) ions are involved in many vital physiological functions. Since dietary intake is the only source of minerals for the body, intestinal absorption is essential for normal homeostatic levels. The aim of this study was to characterize the absorption of Ca(2+) as well as Mg(2+) along the gastrointestinal tract at a molecular and functional level. In both humans and mice the Ca(2+) channel transient receptor potential vanilloid subtype 6 (TRPV6) is expressed in the proximal intestinal segments, whereas Mg(2+) channel transient receptor potential melastatin subtype 6 (TRPM6) is expressed in the distal parts of the intestine. A method was established to measure the rate of Mg(2+) absorption from the intestine in a time-dependent manner by use of (25)Mg(2+). In addition, local absorption of Ca(2+) and Mg(2+) in different segments of the intestine of mice was determined by using surgically implanted intestinal cannulas. By these methods, it was demonstrated that intestinal absorption of Mg(2+) is regulated by dietary needs in a vitamin D-independent manner. Also, it was shown that at low luminal concentrations, favoring transcellular absorption, Ca(2+) transport mainly takes place in the proximal segments of the intestine, whereas Mg(2+) absorption predominantly occurs in the distal part of the gastrointestinal tract. Vitamin D treatment of mice increased serum Mg(2+) levels and 24-h urinary Mg(2+) excretion, but not intestinal absorption of (25)Mg(2+). Segmental cannulation of the intestine and time-dependent absorption studies using (25)Mg(2+) provide new ways to study intestinal Mg(2+) absorption.

The effect of shiga toxin on weibel-palade bodies in primary human endothelial cells.

BACKGROUND/AIMS: Diarrhea-associated hemolytic uremic syndrome is associated with the presence of Shiga toxin (Stx1, Stx2 and several variants) in the circulation. The aim of this study is to examine the possible triggering effect of Stx1 on the exocytosis of Weibel-Palade bodies (WPbs). METHODS: Cultured human umbilical venous endothelial cells (HUVECs) and glomerular microvascular endothelial cells (GMVECs) were stimulated by thrombin and Stx1 in both static and flowing conditions. The amount of secreted von Willebrand factor (VWF) in the supernatant as well as the remaining intracellular fraction was determined. RESULTS: In HUVECs and in 2 out of 4 GMVECs, the stimulation of Stx1 in flow at 1 dyne/cm(2) resulted in a decrease of intracellular VWF. This is contrary to the results of Stx1 applied in static conditions. At a higher flow rate of 5 dyne/cm(2), no effect in GMVECs was observed. CONCLUSION: Stx1 can contribute, via an effect on WPbs, to the exocytosis of WPbs in flow conditions in HUVECs and probably in GMVECs. This results in the release of VWF, suggesting an initiating role of the coagulation system in the pathogenesis.

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.

Evaluation of hypomagnesemia: lessons from disorders of tubular transport.

Hypomagnesemia is a highly prevalent clinical condition affecting a large number of hospitalized patients. A decrease in systemic magnesium concentration may lead to impaired function of both neurologic and cardiovascular systems. The kidney has a pivotal role in magnesium handling by adjusting the urinary excretion of this ion in order to maintain systemic concentrations within a narrow range. As such, the cause of hypomagnesemia can be related to increments in the renal excretion of this cation. Many hypomagnesemic disorders also have characteristic changes in the renal reabsorptive capacity for other electrolytes, leading to symptoms that sometimes obscure the clinical presentation. For instance, changes in serum calcium concentration or its urinary excretion can aid in determining the underlying cause. Moreover, hypokalemia due to renal potassium losses often is associated with hypomagnesemia. Genetic defects in pathways controlling renal electrolyte transport impose the hypomagnesemic condition by facilitating renal losses. The discovery of the causative genes has greatly increased our understanding of how magnesium is transported by the kidney. Such knowledge is integral for the continued improvement of patient care with respect to bettering therapies and diagnosis.

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.