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.

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.

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.

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.

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.

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.

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.

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.

No evidence of hearing loss in pseudohypoaldosteronism type 1 patients.

CONCLUSIONS: The fact that pseudohypoaldosteronism type 1 (PHA-1) patients with a defect in the alpha subunit of epithelial sodium channels (ENaC) in the cochlea have normal hearing suggests compensation by alternative sodium transport mechanisms. Consequently, hearing loss due to defective cochlear transmembrane serine protease TMPRSS3 activity is likely to be related to its effect on proneurotrophin cleavage, indicating an action on neurological components of hearing. The normal hearing of PHA-1 patients with affected mineralocorticoid receptors, together with experimental results in animals, indicates that the mineralocorticoid aldosterone is not the most crucial regulator of sodium transport in the cochlea. OBJECTIVE: Profound hearing loss has been observed in patients with a defect in transmembrane serine protease TMPRSS3, the presumed activator of ENaCs. Renal ENaCs and their regulators, such as the mineralocorticoid receptors, are present in the cochlear structures involved in hearing. The aim of this study was to investigate whether PHA-1 patients with defects in these channels or regulators suffer from hearing impairment. MATERIAL AND METHODS: Pure-tone audiometry was performed in four cases with PHA-1 due to mutations in alphaENaC (n=2) or mineralocorticoid receptor (n=2). RESULTS: All examined cases had normal hearing at all tested frequencies.

Sodium sieving in children.

Sodium sieving is a consequence of dissociation between the amount of water and sodium transported over the peritoneal membrane. This dissociation occurs in the presence of aquaporin-mediated water transport. Sieving of sodium can be used as a rough measure for aquaporin-mediated water transport. Icodextrin contains glucose polymers, inducing ultrafiltration by colloid osmosis. Therefore, aquaporins play a minor role in ultrafiltration, which is confirmed by the absence of sodium sieving. Icodextrin is very suitable for the daytime dwell in children on a nightly intermittent peritoneal dialysis regimen. Ultrafiltration obtained with icodextrin is similar to ultrafiltration obtained with 3.86% glucose after a 12-hour dwell. When using icodextrin in children, it is also confirmed by the absence of sodium sieving that the aquaporins play a minor role in ultrafiltration.

Evaluation of intraperitoneal pressure and the effect of different osmotic agents on intraperitoneal pressure in children.

OBJECTIVES: To establish intraperitoneal pressure (IPP) in a relatively large pediatric study group and to study the effects of a 3.86% glucose solution and a 7.5% icodextrin solution on IPP during a 4-hour dwell. DESIGN: IPP was measured with the patient in a supine position. The intraperitoneal volume (IPV) was 1200 mL/m2 with a 1.36% glucose solution. The influence of dialysis solutions was obtained by performing two 4-hour peritoneal equilibration tests (PETs) with 3.86% glucose and 7.5% icodextrin as test solution, using an IPV of 1200 mL/m2 and dextran 70 as volume marker. IPP was measured at two consecutive time points (t = 0 and t = 240 minutes). Transcapillary ultrafiltration, net ultrafiltration, and marker clearance were calculated. PATIENTS: IPP was established in 30 patients with median age of 4.5 years (range 1.0 - 14.9 years). Influence of dialysis solutions on IPP was studied in 9 children with median age of 4.2 years (range 1.7 - 10.9 years) and median treatment period of 12 months (range 5.6 - 122.3 months). RESULTS: Mean IPP was 12.0 +/- 6.5 cm H2O. Significant relations were found between the change in IPP and transcapillary ultrafiltration and body surface area during the PET with 3.86% glucose. No relations were seen during the PET with icodextrin. CONCLUSIONS: IPP was established in a large pediatric study group and was similar to previously published values of IPP in a small number of patients. Differences in fluid kinetics have different effects on the change in IPP during a 4-hour dwell period.

Renal transplantation in patients with hemolytic uremic syndrome: high rate of recurrence and increased incidence of acute rejections.

BACKGROUND: The reported outcome of renal transplantation in patients with the hemolytic uremic syndrome (HUS) varies greatly, probably related to the diverse causes of HUS. In this single-center retrospective study, we have analyzed the recurrence rate, the incidence of acute rejections, and graft survival in patients suffering from adult-onset and childhood-onset HUS. METHODS: The medical records of 35 patients with end-stage renal disease caused by HUS, who received 50 renal allografts, were reviewed. A definite recurrence of HUS was diagnosed if both clinical and histologic signs of thrombotic microangiopathy (TMA) were present in the absence of any endovasculitis. If there were signs of mild endovasculitis, a probable recurrence was diagnosed. RESULTS: After first renal transplantation, 0 definite and 1 (6%) probable recurrence occurred in 18 patients with childhood-onset HUS, as opposed to 7 (41%) definite and 3 (18%) probable recurrences in 17 adult-onset HUS patients (odds ratio [OR], 13.4; 95% confidence interval [CI], 1.7-105.7). In the latter patients, early use of cyclosporine A increased the risk for recurrence. The incidence of acute rejections was increased compared with matched controls (OR, 1.52; 95% CI, 1.05-2.19 for adult-onset HUS and OR, 1.88; 95% CI, 1.34-2.62 for childhood-onset HUS). One-year graft survival in adult-onset HUS was poor (29%), whereas 1-year graft survival in childhood-onset HUS was comparable to matched controls. CONCLUSIONS: In adult-onset HUS, the recurrence rate and the incidence of acute rejections are high, resulting in a detrimental graft survival. In childhood-onset HUS, the recurrence rate is low, but the posttransplantation course is complicated by an increased incidence of acute rejections.

Dominant isolated renal magnesium loss is caused by misrouting of the Na+,K+-ATPase gamma-subunit.

Hereditary primary hypomagnesemia comprises a clinically and genetically heterogeneous group of disorders in which hypomagnesemia is due to either renal or intestinal Mg(2+) wasting. These disorders share the general symptoms of hypomagnesemia, tetany and epileptiformic convulsions, and often include secondary or associated disturbances in calcium excretion. In a large Dutch family with autosomal dominant renal hypomagnesemia, associated with hypocalciuria, we mapped the disease locus to a 5.6-cM region on chromosome 11q23. After candidate screening, we identified a heterozygous mutation in the FXYD2 gene, encoding the Na(+),K(+)-ATPase gamma-subunit, cosegregating with the patients of this family, which was not found in 132 control chromosomes. The mutation leads to a G41R substitution, introducing a charged amino acid residue in the predicted transmembrane region of the gamma-subunit protein. Expression studies in insect Sf9 and COS-1 cells showed that the mutant gamma-subunit protein was incorrectly routed and accumulated in perinuclear structures. In addition to disturbed routing of the G41R mutant, Western blot analysis of Xenopus oocytes expressing wild-type or mutant gamma-subunit showed mutant gamma-subunit lacking a posttranslational modification. Finally, we investigated two individuals lacking one copy of the FXYD2 gene and found their serum Mg(2+) levels to be within the normal range. We conclude that the arrest of mutant gamma-subunit in distinct intracellular structures is associated with aberrant posttranslational processing and that the G41R mutation causes dominant renal hypomagnesemia associated with hypocalciuria through a dominant negative mechanism.

Intravenous iron supplementation in children on hemodialysis.

BACKGROUND: Children with end-stage renal disease (ESRD) on hemodialysis (HD) are often absolute or functional iron deficient. There is little experience in treating these children with intravenous (i.v.) iron-sucrose. In this prospective study, different i.v. iron-sucrose doses were tested in children with ESRD on HD and the effect on iron status measured. METHODS: Fourteen patients were divided into three groups according to their actual iron status. Group A--iron deficient (ferritin (F)<100 microg/L, or F 100-400 microg/L and transferrin saturation (TSAT)<20%). These patients were treated with i.v. iron-sucrose 3 mg/kg/dialysis. Group B--iron-replete (F 100-400 microg/L and TSAT> or =20%, or TSAT>50%). These patients received 0.3 mg/kg/dialysis iron-sucrose. Group C--possible iron-overloaded (F>400 microg/L). These patients were not treated with iron. RESULTS: Group A--3 mg/kg/dialysis of iron-sucrose resulted in a major increase in F, indicating possible iron overload. Therefore, the iron-deficient patients received 1 mg/kg/dialysis iron-sucrose during 22 periods of 2-14 (mean 5) weeks: the median F increased from 186 to 343 microg/L (p<0.001). Group B--0.3 mg/kg/dialysis iron-sucrose resulted in adequate iron levels during 22 periods of 2-60 (mean 9) weeks. CONCLUSION: In children, 3 mg/kg/dialysis iron-sucrose complex results in a possible iron overload. Dosage of 1 mg/kg/dialysis and 0.3 mg/kg/dialysis seem adequate for correction and maintenance therapy respectively.

Compartment syndrome, rhabdomyolysis and risk of acute renal failure as complications of the lithotomy position.

Compartment syndrome, rhabdomyolysis and the risk of acute renal failure are potential complications of the lithotomy position. A six-year-old girl is described who developed a compartment syndrome with rhabdomyolysis after prolonged surgery in the lithotomy position. This complication occurred three times over ten years in our hospital. Rhabdomyolysis may induce acute renal failure. The pathogenesis of rhabdomyolysis--induced renal failure has not yet been elucidated. However, forced diuresis by intravenous administration of mannitol and furosemide can prevent acute renal failure.

Is serum cystatin C the marker of choice to predict glomerular filtration rate in paediatric patients?

BACKGROUND: It has been suggested that serum cystatin C (cyst-C) concentration provides a better indication of changes in glomerular filtration rate (GFR) than does serum creatinine concentration. METHODS: Because of conflicting results as to the usefulness of cyst-C, we compared the GFRs calculated from serum cyst-C, inulin clearance and endogenous creatinine clearance in children. GFRs calculated from cystatin concentration, inulin clearance following a single injection and endogenous creatinine clearance using Jaffé and enzymic methods were compared in 66 children (1.3-21.9 years) with a variety of renal disorders. Receiver operating curve analysis was used to determine the cut-off value that would give the best discrimination between normal and decreased GFR. RESULTS: The serum cyst-C concentration ranged from 0.66 to 7.61 mg/L (median 1.94). Serum creatinine Jaffé concentration (creat-J) ranged from 38 to 871 micro mol/L (median 105) and creatinine enzymatic concentration (creat-E) ranged from 28 to 862 micro mol/L (median 126). The linear correlation coefficient (R) of 1/cyst-C versus GFR (R = 0.937) did not differ from either that of 1/creat-J versus GFR (R = 0.918) or that of 1/creat-E versus GFR (R = 0.901). These coefficients had overlapping confidence intervals. The areas under the curve for cyst-C, creat-J and creat-E were 0.967, 0.977 and 0.924, respectively, and were not significantly different from each other. For cyst-C, the optimal cut-off was 1.1 mg/L. CONCLUSIONS: Serum cyst-C is equivalent to creat-J and creat-E as a marker for estimating the GFR in the paediatric population studied.

Fibrin glue used successfully in peritoneal dialysis catheter leakage in children.

BACKGROUND: Acute renal failure in infants and small children is generally treated with peritoneal dialysis (PD). Dialysis has to be started immediately after catheter implantation. Early dialysate leakage can complicate the effectiveness of dialysis. Fibrin glue applied to the external part of the tunnel may stop dialysate leakage and eliminate the need for surgical intervention. The use of fibrin glue in the treatment of PD catheter leakage in children was studied. METHODS: Fibrin glue was used in 8 children (age 0.8 - 57 months) on PD in whom dialysate leakage was seen during the first 24 to 48 hours after catheter insertion. The dialysis volume initially administered was 20 mL/kg body weight. Fibrin glue (1 mL) was applied to the external part of the subcutaneous catheter tunnel through the exit site, as close to the cuff as possible. The occurrence of dialysate leakage and complications such as exit-site or tunnel infection and peritonitis were evaluated. RESULTS: Nine single-cuff straight Tenckhoff catheters were implanted in 8 children. In 5 cases, no subcutaneous tunnel was created. One child had catheter replacement due to obstruction of the catheter; on both occasions, catheter leakage was seen and treated with fibrin glue. In all 8 patients, no relapse of dialysate leakage was seen after application of the fibrin glue. During the time of PD, exit-site infections, tunnel infections, and peritonitis did not occur. CONCLUSION: Fibrin glue is a successful, simple, and safe substance for the treatment of peritoneal dialysate leakage in infants and small children with acute renal failure treated with PD.

Serial plasma concentrations of atrial natriuretic peptide, plasma renin activity, aldosterone, and antidiuretic hormone in neonates on extracorporeal membrane oxygenation.

To obtain information on water and salt regulating hormones and volume homeostasis during neonatal extracorporeal membrane oxygenation (ECMO), serial determinations of atrial natriuretic peptide (ANP), plasma renin activity (PRA), aldosterone (Aldo), antidiuretic hormone (ADH), colloid-osmotic pressure (COP), osmolality (Osmol), and central venous pressure (CVP) before, during, and after neonatal ECMO in 10 neonates with meconium aspiration syndrome (MAS) were carried out. Mean gestational ages and birth weights were 41(+3) weeks (39(+6) - 42(+4)) and 4,063 gm (3,500-4700), respectively; mean age at start and duration of ECMO 29.3 (14-69) and 152.6 hr (92-267), respectively. Plasma ANP (mean +/- SD) was 67.8+/-69.1 pmol/L before, decreased to 33.3+/-22.1 (not significant) pmol/L during, and significantly increased to 274.6+/-131.8 pmol/L after ECMO (p < 0.05). ANP correlated positively with CVP (r = 0.63; p < 0.001). Pre-ECMO PRA, Aldo, and ADH were comparable to those described earlier in normal neonates, decreased during (p < 0.001 for Aldo; p < 0.05 for PRA and ADH) and either remained elevated (PRA, p < 0.001; Aldo, p < 0.05) or decreased (ADH) after ECMO. COP and Osmol remained unchanged. Neonatal ECMO for MAS is characterized by circulatory and osmotic equilibrium. It is suggested that circulating volume contracts during and expands after neonatal ECMO for MAS.