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03/2012; , ISBN: 978-953-51-0245-8
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ABSTRACT: 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.
Cell and Tissue Research 11/2009; 339(2):449-57. · 3.11 Impact Factor
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Nephrology Dialysis Transplantation 08/2008; 23(9):2719-22. · 3.40 Impact Factor
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ABSTRACT: 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.
Pediatric Research 07/2008; 64(5):495-7. · 2.70 Impact Factor
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ABSTRACT: Shiga toxin (Stx) is the main pathogenic factor in the haemolytic-uraemic syndrome (HUS). Stx damages the renal endothelium, which leads to inflammation and coagulation. Endothelial heparan sulfate proteoglycans (HSPG), and heparan sulfate in particular, play an important role in the inflammatory process by acting as a ligand for l-selectin. Furthermore, leukocytes are able to interact with chemokines bound to HSPG (examples are IL-8, RANTES and MCP-1). This leads to an activation of integrins on leukocytes and results in more stable leukocyte-endothelial wall adhesion. In this study, we have evaluated the effect of a subtoxic dose of Stx1 and Stx2 on the HSPG and its role in adhesion of leukocytes.
Primary human umbilical venous endothelial cells (HUVEC) and primary human glomerular microvascular endothelial cells (GMVEC) were incubated for 24 h with a subtoxic dose of Stx1 or Stx2. Then, cells were treated with heparan sulfate-degrading enzyme heparitinase I or left untreated, followed by determination of binding leukocytes to endothelial cells in a parallel plate flow chamber.
In both cell types, Stx increased the amount of firmly adherent leukocytes. After removal of endothelial heparan sulfate, the number of adhering leukocytes decreased.
HSPG have a distinctive role in adhesion of leukocytes to endothelial cells stimulated by a subtoxic dose of Stx.
Nephrology Dialysis Transplantation 06/2008; 23(10):3091-5. · 3.40 Impact Factor
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ABSTRACT: Focal segmental glomerulosclerosis (FSGS) is a major cause of steroid-resistant nephrotic syndrome in childhood with a central role for the podocytes in the pathogenesis. Mutated proteins expressed in podocytes cause proteinuria. The role of combined gene defects in the development of FSGS is less clear.
We analysed seven podocyte genes known to cause proteinuria and FSGS in a group of 19 non-familial childhood-onset steroid-resistant FSGS patients. These genes include NPHS1, NPHS2, ACTN4, CD2AP, WT-1, TRPC6 and PLCE1. We also screened for the mitochondrial A3243G DNA transition associated with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), and occasionally FSGS.
No mutations were found in the ACTN4 and TRPC6 genes, and no mitochondrial A3243G DNA transition was found in our group of patients. Two patients showed mutations in the CD2AP gene, one combined with an NPHS2 mutation. A tri-allelic hit was found in a patient carrying compound heterozygous NPHS2 mutations and a heterozygous NPHS1 mutation. In another patient a de novo WT-1 mutation was found combined with a heterozygous NPHS1 mutation, and finally two patients showed three heterozygous PLCE1 mutations.
In our rather small group of 19 steroid-resistant FSGS patients, we found 11 mutations in podocyte genes in 6 patients. In four of them the found mutations could explain the pathology. Our data suggest that combined gene defects in podocyte genes may play a role in the development of FSGS.
Nephrology Dialysis Transplantation 05/2008; 23(10):3146-51. · 3.40 Impact Factor
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ABSTRACT: A genetic predisposition involving complement regulatory genes has become evident in some patients with atypical HUS. In this paper, a patient with a heterozygous missense mutation in factor I (IF) is described. Although the serum level of IF was normal, a mild functional defect in the alternative pathway of complement could be demonstrated in the affected members of the family. After an episode of atypical HUS, chronic renal insufficiency started at the age of 15 months. Recurrence of HUS, with loss of the renal transplant, occurred twice in this patient. The recurrence of HUS in the graft was not reflected by haematological abnormalities (haemolysis, thrombocytopenia). One additional transplant was lost due to arterial thrombosis of the renal artery. This report confirms the gloomy outcome of renal transplants in patients with an IF deficiency. New therapies should be evaluated in these patients.
Pediatric Nephrology 04/2007; 22(3):371-5. · 2.52 Impact Factor
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ABSTRACT: Fungal peritonitis is a rare but serious complication in children on peritoneal dialysis (PD). In this study, risk factors were evaluated, and therapeutic measures were reviewed. A retrospective, multi-centre study was performed in 159 Dutch paediatric PD patients, between 1980 and 2005 (3,573 months). All peritonitis episodes were reviewed. Fungal peritonitis episodes were evaluated based on possible risk factors and treatment strategy. A total of 321 episodes of peritonitis occurred, with 9 cases of fungal peritonitis (2.9%). Candida peritonitis occurred most frequently (78%). Seven patients (78%) had used antibiotics in the prior month. Fungal peritonitis patients had a higher previous bacterial peritonitis rate compared to the total study population (0.13 versus 0.09 episodes/patient*month), with twice as many gram negative organisms. In all fungal peritonitis patients, the PD catheter was removed. In four patients restart on PD was possible. Fungal peritonitis is a rare complication of PD in children, but is associated with high technique failure. The most important risk factors are a high bacterial peritonitis rate, prior use of antibiotics, and previous bacterial peritonitis with gram negative organisms. The PD catheter should be removed early, but in children, peritoneal lavage with fluconazole before removal may be useful to prevent technique failure.
Pediatric Nephrology 03/2007; 22(2):288-93. · 2.52 Impact Factor
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ABSTRACT: Acute renal failure hallmarks the pathogenesis of the epidemic form of hemolytic uremic syndrome (D+HUS), which is caused by E. coli strains that produce Shiga-like toxin (Stx). In this study, we investigated the influence of Stx-1 on nitric oxide (NO) production by human glomerular microvascular endothelial cells (GMVEC) and human mesangial cells. NO synthesis by human mesangial cells is in the micromolar range and that of GMVEC in the picomolar range. Stx-1 reduced NO production in non-stimulated GMVEC (5 nmol/l Stx-1 required) without inhibition of protein synthesis. In non-stimulated and TNFalpha-pretreated mesangial cells, NO production was reduced with a maximal reduction at 10 fmol/l shiga toxin. The cellular iNOS antigen content in mesangial cells was reduced in a concentration-dependent way (10 fmol/l-100 pmol/l), while partial inhibition of protein synthesis required 10 nmol/l Stx-1 in these cells. Our in vitro data suggest that Stx may reduce NO synthesis during the course of HUS development, contributing to the aggravation of the thrombotic microangiopathy and renal failure as observed in HUS.
Pediatric Nephrology 01/2007; 21(12):1815-23. · 2.52 Impact Factor
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Acta Paediatrica 04/2006; 95(3):379-80. · 2.07 Impact Factor
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ABSTRACT: Cystinosis is an autosomal recessive disorder, caused by mutations of the lysosomal cystine carrier cystinosin, encoded by the CTNS gene (17p13). The concomitant intralysosomal cystine accumulation leads to multi-organ damage, with kidneys being the first affected. Altered mitochondrial oxidative phosphorylation has been demonstrated in animal proximal tubules loaded with cystine dimethyl ester, mimicking cystine accumulation in cystinosis, but has not been confirmed in cells of patients with cystinosis. Furthermore, the link between cystine accumulation and mitochondrial damage is also missing. We hypothesized that cytosolic cysteine deficiency resulting in intracellular glutathione (GSH) shortage might be involved in cellular dysfunction in cystinosis.
Components of the gamma-glutamyl cycle were measured in cultured skin fibroblasts (n = 9) and polymorphonuclear (PMN) leukocytes (n = 15) derived from patients with cystinosis and compared with the values in cultured fibroblasts (n = 9) and PMN cells (n = 18) of healthy controls.
Cystine content in cystinotic fibroblasts and PMN cells was significantly elevated compared with the controls, consistent with the lysosomal cystine accumulation in these cells. Although no reduction of total intracellular GSH content was found in cystinotic cells, it inversely correlated with cystine levels. Furthermore, GSH disulfide (GSSG) was elevated in cystinotic cells, resulting in an increased GSSG/total GSH (%) ratio. No relationship between intracellular cystine and GSH was found in control fibroblasts and PMN cells.
An elevated GSSG/total GSH (%) ratio might indicate increased oxidative stress present in cystinotic cells. Inverse correlation between cystine accumulation and intracellular GSH content indicates that under stress conditions such as intensive energy demand or increased oxidative insult, cystinotic cells may be more prone to GSH depletion.
Nephrology Dialysis Transplantation 10/2005; 20(9):1828-32. · 3.40 Impact Factor
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Clinical Chemistry 10/2004; 50(9):1686-8. · 7.91 Impact Factor
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ABSTRACT: The pathogenesis of hemolytic uremic syndrome (D+ HUS) is characterized by endothelial damage of glomeruli and tubules within the kidney. In several other diseases in which glomerular endothelial damage occurs, elevated serum levels of vascular endothelial growth factor (VEGF) have been reported. VEGF is involved in angiogenesis, permeabilization of blood vessel endothelium, and wound repair. In this study we evaluated VEGF levels in the serum of 40 D+ HUS patients in the acute phase and during the course of the disease. VEGF levels were measured using a double-sandwich ELISA. Indirect immunohistochemistry was performed for the detection of VEGF in renal biopsy material of 3 HUS patients. Significantly elevated VEGF levels were found in HUS patients compared with controls in both serum ( P<0.001) and plasma ( P<0.05). A significant relationship was found between VEGF levels and severity of the disease according to the classification of Gianantonio ( P<0.05). Levels of VEGF in blood increased during the 2nd and 3rd week after HUS was diagnosed. Immunohistochemistry of renal biopsy material showed increased levels of the receptors for VEGF in the glomeruli. During the course of HUS, plasma VEGF levels increase and the increase is dependent on the severity of the disease. This is probably associated with the repair process.
Pediatric Nephrology 08/2004; 19(7):754-60. · 2.52 Impact Factor
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ABSTRACT: Between 2 and 4 h after miction the morphology of urinary erythrocytes has changed, clouding the distinction between glomerular and non-glomerular bleeding in pediatric patients. Glomerular bleeding is characterized by microscopically visible alteration in urinary erythrocytes due to glomerular disease. Fixation by Cellfix, a formaldehyde-based fixative, allows the preservation of the morphology for at least 24 h and can be recommended for clinical practice. In our experience, thiomersal was not effective for preservation of the morphology of erythrocytes.
Pediatric Nephrology 08/2003; 18(7):665-6. · 2.52 Impact Factor
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ABSTRACT: The pentad of thrombocytopenia, hemolytic anemia, mild renal dysfunction, neurologic signs, and fever, classically characterizes the syndrome of thrombotic thrombocytopenic purpura (TTP). TTP usually occurs in adults as an acquired form but a congenital form in children has also been described. In the latter case, the initial presentation is often with neonatal jaundice and thrombocytopenia. The disorder may subsequently take a relapsing course. Deficiency of a recently identified novel metalloprotease, the von Willebrand factor (vWF)-cleaving protease, originating from mutations in the ADAMTS13 gene plays a major role in the development of TTP.
Blood for DNA analysis was collected from six unrelated TTP families, consisting of nine patients from four different countries, and was screened for mutations in the ADAMTS13 gene. This gene spans 29 exons encompassing approximately 37 kb. Conventional techniques of DNA extraction, polymerase chain reaction (PCR), and direct cycle sequencing were used.
Eight novel ADAMTS13 mutations are presented. Half of the total number of mutant ADAMTS13 alleles are amino acid substitutions. The disease-causing mutations are spread over the gene. The pathogenicity of the individual mutations is based upon their predicted effect on the ADAMTS13 protein and segregation in family members. Although most of the patients (seven out of nine) had symptoms during the neonatal period, they were in a remarkably good condition. Only one of the nine patients had a decreased glomerular filtration rate (GFR) with proteinuria and hematuria. Another patient had epileptic seizures.
We confirm that deficiency of ADAMTS13 is a molecular mechanism responsible for familial TTP. An early diagnosis allows prophylactic treatment with fresh plasma infusions.
Kidney International 07/2003; 63(6):1995-9. · 6.61 Impact Factor
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ABSTRACT: Mutation analysis and clinical implications of von Willebrand factor–cleaving protease deficiency.Background The pentad of thrombocytopenia, hemolytic anemia, mild renal dysfunction, neurologic signs, and fever, classically characterizes the syndrome of thrombotic thrombocytopenic purpura (TTP). TTP usually occurs in adults as an acquired form but a congenital form in children has also been described. In the latter case, the initial presentation is often with neonatal jaundice and thrombocytopenia. The disorder may subsequently take a relapsing course. Deficiency of a recently identified novel metalloprotease, the von Willebrand factor (vWF)–cleaving protease, originating from mutations in the ADAMTS13 gene plays a major role in the development of TTP.
Kidney International 05/2003; 63(6):1995-1999. · 6.61 Impact Factor
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ABSTRACT: The diarrhea-associated form of the hemolytic uremic syndrome (D+ HUS) is characterized by a triad of symptoms, namely thrombocytopenia, hemolytic anemia, and acute renal failure. Histopathological studies of patients with D+ HUS show microthrombi in arterioles and glomeruli of the kidney. Recently, it was suggested that antiphospholipid antibodies might play a pathogenic role in D+ HUS. However, an epiphenomenon could not be excluded. In this study we investigated the relationship between antiphospholipid antibodies and clinical symptoms in 22 patients with the classical form of HUS (D+ HUS). The first sample was obtained on the day of admission. The next samples were taken on day 7 and 14. We measured anticardiolipin (aCL) antibodies (IgM, IgA, and IgG) in the samples using an ELISA. A significant increase in IgM (60%) and IgG (41%) aCL antibodies was seen in patients versus controls. No relationship between aCL antibody levels and severity of renal failure could be demonstrated. These data suggest that antiphospholipid antibodies are increased, but have not been shown to have a role in the pathogenesis of the microangiopathy seen in D+HUS.
Pediatric Nephrology 01/2003; 17(12):1042-6. · 2.52 Impact Factor
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Carla Zoja,
Stefania Angioletti,
Roberta Donadelli,
Cristina Zanchi,
Susanna Tomasoni,
Elena Binda,
Barbara Imberti,
Maroeska te Loo, Leo Monnens,
Giuseppe Remuzzi,
Marina Morigi
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ABSTRACT: Shiga toxin (Stx)-producing E. coli is a causative agent of the epidemic form of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Endothelial injury and leukocyte activation are instrumental to the development of microangiopathic lesions. To obtain more insight into the mechanisms favoring endothelium-leukocyte interaction, we studied (1) the effect of Stx-2 on leukocyte adhesion and transmigration in human endothelial cells under flow; (2) the effect of Stx-2 on endothelial expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) and their functional role in the adhesive phenomena; and (3) the role of nuclear factor-kappaB (NF-kappaB) in endothelial chemokine expression.
For adhesion experiments, human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells (GEC) were incubated for 24 hours with Stx-2 (25 pmol/L), with or without anti-IL-8 or MCP-1 antibodies, and then exposed to leukocyte suspension under flow (1.5 dynes/cm2). IL-8 and MCP-1 expression was evaluated in Stx-2 treated endothelial cells (6 hours) by Northern blot. NF-kappaB activity was assessed by electrophoretic mobility shift assay. The role of NF-kappaB in Stx-induced chemokines was evaluated by transfecting HUVEC with an adenovirus coding for IkappaBalpha.
Stx-2 significantly enhanced the number of leukocytes that adhered and then migrated across the endothelium. Stx-2 increased the expression of IL-8 and MCP-1, which was preceded by NF-kappaB activation. Blocking of endothelial IL-8 and MCP-1 with corresponding antibodies significantly inhibited Stx-induced leukocyte adhesion and migration either in HUVEC or GEC. Adenovirus-mediated gene transfer of IkappaBalpha down-regulated IL-8 and MCP-1 mRNA and also inhibited the adhesion and transmigration of leukocytes in Stx-treated HUVEC.
Stx-2 via a transcriptional activation mechanism specifically mediated by NF-kappaB up-regulates endothelial MCP-1 and IL-8. Both chemokines are important modulators of leukocyte adhesion and transmigration under flow. These findings might be relevant to understand the nature of microvascular lesions in HUS and open future perspectives for better treatment of microvascular thrombosis.
Kidney International 10/2002; 62(3):846-56. · 6.61 Impact Factor
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Carla Zoja,
Stefania Angioletti,
Roberta Donadelli,
Cristina Zanchi,
Susanna Tomasoni,
Elena Binda,
Barbara Imberti,
Maroeska Te Loo, Leo Monnens,
Giuseppe Remuzzi,
Marina Morigi
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ABSTRACT: Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-B dependent up-regulation of IL-8 and MCP-1.Background Shiga toxin (Stx)-producing E. coli is a causative agent of the epidemic form of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Endothelial injury and leukocyte activation are instrumental to the development of microangiopathic lesions. To obtain more insight into the mechanisms favoring endothelium-leukocyte interaction, we studied (1) the effect of Stx-2 on leukocyte adhesion and transmigration in human endothelial cells under flow; (2) the effect of Stx-2 on endothelial expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) and their functional role in the adhesive phenomena; and (3) the role of nuclear factor-B (NF-B) in endothelial chemokine expression.
Kidney International 08/2002; 62(3):846-856. · 6.61 Impact Factor
