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Mitochondrial functions have a major impact on T-cell functionality. In this study we characterized whether mitochondrial function in the neonatal T-cells differs from that in the adult T-cells during short T-cell activation. Methods:We used fow cytometry methods to test mitochondrial mass and to monitor mitochondrial Ca2+levels, mitochondrial potential and superoxide generation in parallel with cytoplasmic Ca2+levels during phythohaemagglutinine-induced activation of CD4+ and CD8+ T-cells of 12 term neonates and 11 healthy adults. Results:Baseline mitochondrial mass of CD4+ and CD8+ cells was lower in the neonate than in the adult. In comparison with the adult, neonatal resting CD4+ T-cells had lower cytoplasmic Ca2+levels and this was associated with normal activation induced Ca2+-response. During short-term activation cytoplasmic Ca2+-response was lower in neonatal than in adult CD8+ T-cells. Mitochondrial Ca2+uptake was increased in CD4+ neonatal T cells while it decreased in CD8+ T-cells. Mitochondrial depolarization was increased in CD4+ and decreased in CD8+ neonatal T-cells compared to adults. Superoxide generation was higher and equal in neonatal CD4+ and CD8+ cells, respectively, compared to the adult ones. Conclusion:Our data suggest that neonatal T-cells exhibit marked differences in mitochondrial function and superoxide generation compared to adult T-cells.

Previous experimental data suggest that steroids might have protective effects during hypoxic/ischemic injury of various organs. In this study, the association between dexamethason (Dexa) treatment and the anti-apoptotic SGK-1 was tested in ischemic renal injury. In vitro, HK-2 cells were exposed to 24 h hypoxia, and the effect of Dexa incubation on SGK-1 expression / activation and on cell death was studied. In an in vivorat model of unilateral renal IR, animals were treated with Dexa, and serum renal function parameters, tissue injury and SGK-1 expression and localization were examined after different reperfusion times (2 h, 4 h and 24 h). Dexa at a dose of 2 mg/L exerted a protective effect on cell survival assessed by LDH release and vital staining paralleled by marked up-regulation of SGK-1. In rats, 2 mg/kg Dexa treatment 24 h prior to ischemia resulted in less severe tissue injury and ameliorated urea nitrogen levels 24 h after reperfusion. Furthermore, SGK-1 expression and phosphorylation were higher in Dexa animals demonstrated by Western blot and immunofluorescence technique. Our results provide novel data on the signalling mechanism of Dexa under hypoxia / ischemia and further support that Dexa emerges as an attractive pharmacological agent for the prevention of ischemic injury.

Flow cytometry enables the sequential determination of calcium levels in millions of stimulated lymphocytes over a short period of time. Current algorithms available are not suitable for the statistical analysis of this large amount of data. The authors aimed to develop a robust algorithm that fits a function to median values of measured data and provides an opportunity for statistical comparison between different calcium‐flux measurements. The alteration of calcium signal was monitored in CD4+ cells loaded with calcium binding fluorescent dyes and stimulated with phytohemagglutinin; the alteration of calcium signal was monitored for 10 minutes. The authors also reanalyzed published calcium‐flux data of CD3+ cells and Jurkat cells stimulated with different concentrations of anti‐CD3 and thapsigargin. The authors fitted different functions to the medians of data per time unit and identified hormesis function as the best fitting one. On the basis of the optimally fitting function, the authors calculated the most relevant biological descriptors such as starting value, peak, time to reach the maximum, and time to reach 50% of maximum before and after the peak. Statistically significant differences in cell activation kinetics at different stimulatory concentrations were also demonstrated. This approach enables us to characterize the kinetics and distribution of calcium‐flux data derived by flow cytometry and may be a reliable tool for the characterization of lymphocyte activation (for details see: http://calciumflux.intralab.eu). © 2007 International Society for Analytical Cytology

Abstract.: Higher erythrocyte sodium-lithium countertransport activity (SLC) is implicated in the development of diabetic nephropathy. Altered glucose homeostasis and genetic susceptibility are claimed to play a role in the elevation of SLC. We aimed to test whether metabolic control or the genetic variants of G protein beta 3 (Gb3) subunits determine SLC and other erythrocyte transport activities in complication-free stage of type 1 diabetes. A total of 96 complication-free type 1 diabetic children and adolescents were enrolled. SLC, Na+/K+-ATPase (NAK) and Ca2+-ATPase (CA) were measured by functional assays in erythrocytes. Gb3-C825T polymorphism was determined by PCR-RFLP. Results were related to HbA1c and were compared to those of 97 healthy controls. SLC activity was higher in diabetics (387±146 vs. 280±65 mmol/RBC · hour) and correlated with HbA1c levels (y=0.004x+6.42, r=0.33, n=96, p<0.01). NAK and CA activities were unaltered. The prevalence of 825T allele was similar in the patient and control groups (0.34 vs 0.37) and no differences in enzyme activities were observed between the 825T allele-positive and negative subjects. Although metabolic control correlated with SLC, other membrane functions were not affected. Therefore we hypothesize that the relationship between advanced glycation and SLC elevation is not causative. Rather, a genetic susceptibility for the coexistence of poor metabolic control and higher SLC is more likely. However, the presence of Gb3-C825T variant is not likely to be a risk factor for SLC-elevation and altered metabolic control diabetes.

OBJECTIVE: Recent data have indicated the significance of vitamin D receptor (VDR) polymorphisms in type 1 diabetes mellitus (T1DM). We have studied the association of five known restriction enzyme polymorphisms of the VDR gene in patients with T1DM. DESIGN AND METHODS: One hundred and seven children with T1DM (T1DM for 5 Years; age, 1-14 Years; boys/girls, 57/50; body mass index, 17.0+/-2.3 kg/m(2); haemoglobin A(Ic) (HbA(Ic)), 7.87+/-1.05) and 103 healthy subjects were enrolled. The VDR polymorphisms ApaI, BsmI, FokI, TaqI and Tru9I ("a", "b", "f", "t" and "u" alleles respectively) were investigated. RESULTS: The "t" and "T" alleles miss the Hardy-Weinberg equilibrium (P<0.01) in control and diabetic populations; we therefore excluded this polymorphism from further analysis. We did not find a difference in the allele prevalence in T1DM patients and controls of any of the five polymorphisms. However, when the "b", "a" and "u" alleles were simultaneously compared in girls, there was a significantly higher prevalence in patients with diabetes compared with controls ("b"+"a"+"u" present/absent: healthy, 0/53; diabetic, 13/37; P<0.005). In boys the prevalence of "b"+"a"+"u" genotype was similar in T1DM and controls. CONCLUSIONS: The impact of the "t" allele cannot be investigated in this study population. Not a single VDR polymorphism increases the susceptibility to T1DM. The common presence of the "b", "a" and "u" alleles greatly increases the probability of T1DM in girls.

We developed a new hypothesis claiming that natriuresis of fasting is not only caused by diminished insulin resistance and hyperinsulinaemia with the subsequent reduction of renal sodium retention but it can also be attributed to the function of the leptin–NPY system. Each element of this concept has been substantiated by convincing experimental evidences as follows:

AimTo study bone turnover following renal transplantation using a panel of biochemical markers and to correlate the results with both areal and volumetric bone mineral density (BMD).PatientsA total of 31 patients aged 18.1 years were transplanted 5.4 years before this study. Control patients (n = 31) were age and gender matched.MethodsIn addition to measurement of biochemical markers, BMD was measured by single photon absorptiometry and peripheral quantitative computed tomography on the non-dominant radius.ResultsPatients had reduced glomerular filtration rate, raised concentrations of serum phosphate, serum procollagene type I carboxy terminal propeptide, osteocalcin, and serum procollagene type I cross linked carboxy terminal telopeptide. The differences were still significant if only patients with normal intact parathyroid hormone were considered. BMD single photon absorptiometry Z score for age was significantly decreased. Following standardisation for height the differences were no longer present. With volumetric techniques patients had normal trabecular but decreased cortical and total BMD compared to age matched controls, but there was no difference from height matched controls.ConclusionMarkers of bone turnover are increased following renal transplantation. However, the biochemical analysis did not allow conclusions to be drawn on the bone mineral content. BMD single photon absorptiometry Z score corrected for height and BMD measured by quantitative computed tomography compared to height matched controls were normal in paediatric renal transplantation patients. Height matched controls should be used in both areal and volumetric BMD measurements in states of growth failure.

The effect of low dose (2, 4, and 8 µg/kg per min) dopamine infusion on blood pressure, heart rate and renal function was studied in 18 hypotensive, preterm infants with severe hyaline membrane disease (HMD). Significant dose-related effects found during dopamine infusion were systolic and diastolic blood pressure elevation and diuretic effect, while heart rate increase occurred only with 8 µg/kg per min of the drug. This indicates, that in the preterm neonate, dopamine at low doses has a pronounced effect on the alpha-and dopamine-receptors, while its beta-receptor stimulating activity is minimal. We demonstrated a significantly decreased metabolic clearance rate of dopamine in preterm infants. Thus, beside the differences in the vascular receptors' maturation, the decreased metabolic clearance rate should also be taken into account when explaining the cardiovascular and renal effects of low dose dopamine infusion in these babies. Dopamine was found to be useful in normalizing low arterial blood pressure, in improving impaired peripheral circulation, and in producing a marked diuresis in hypotensive preterm neonates with severe hyaline membrane disease.

Urinary excretion of endothelin was measured by radioimmunoassay in children with diabetes mellitus during severe ketoacidosis and 12 days later when blood pH and blood glucose concentrations were normal. Metabolically stable diabetic children served as controls. Results from apparently healthy children without diabetes mellitus were used as normal values. Renal tubular injury was evaluated by the urinary excretion of the proximal tubular marker a1-microglobulin and the distal tubular marker Tamm-Horsfall protein (THP). During ketoacidosis we detected a decreased glomerular filtration rate associated with highly significant changes in the excretion of a1-microglobulin, indicating proximal tubular damage, and THP, suggesting disturbance of cells of the ascending loop of Henle. The daily excretion of endothelin was unaltered but the ratio of endothelin/creatinine or endothelin excretion/creatinine clearance were significantly enhanced. In conclusion, we could demonstrate that, despite a proximal and distal tubular cell dysfunction, endothelin excretion when related to creatinine clearance may be a consequence of disturbed proximal tubular function or dysfunction of the renal medulla.

In pharmacological doses dopamine (DA) will interact with several endocrine systems and both inhibit (prolactin, thyrotropin) and enhance (renin, angiotensin) hormonal release. In this study we have examined whether DA given to preterm neonates will influence prostaglandin (PG) production. The question is of importance since vasodilatator PGs play a role in postnatal adaptation. We determined the effect of low dose DA infusion on the 24 h urinary PGE2 excretion rate (an index of renal PGE2 synthesis) in preterm infants. Six preterm neonates, with a 24-h requirement of 2 µg/kg per min DA treatment for oedema, moderate oliguria, poor peripheral perfusion and/or mild systemic hypotension were studied on days 2 (Day 1), 3 (Day 2, the day of DA infusion), and 4 (Day 3, DA discontinued) of life. Six preterm infants (control group) that did not require DA infusion were also studied to monitor possible spontaneous changes in the renal PGE2 production on days 2, 3 and 4 of life. In the control group urine output (Uv) and PGE2 excretion rate remained unchanged during the study. In the study group DA administration resulted in nearly two-fold increases in both the Uv (194%) and PGE2 excretion (182%). Urinary PGE2 excretion was, however, closely related to urine flow in both the control infants (Day 1–3) and the study group infants (Day 1–2). Since increased diuresis stimulates renal PGE2 production, our data suggest that the increased PGE2 excretion on Day 2 in the study group was not due to a direct effect of DA on PGE2 synthesis. On Day 3, however, urinary PGE2 excretion in the study group decreased out of proportion to that of the Uv (-66% vs-23%), indicating that discontinuation of the drug infusion directly decreases renal PGE2 synthesis. In conclusion, the findings of the present study indicate that low dose DA does not directly trigger renal PGE2 production in the sick preterm infant.

The effect of volume changes on the plasma concentration of atrial natriuretic peptide (ANP) was investigated. In children with chronic renal failure predialysis plasma ANP was higher as compared to normal children and decreased significantly during hemodialysis. The decrease in plasma ANP correlated with body weight reduction. On the other hand, increase in plasma volume by infusion of albumin in children with nephrotic syndrome caused significant rise in plasma ANP and an increased natriuresis. Our data demonstrate that an expanded extracellular fluid volume and/or plasma volume appears to be a major stimulus for the release in ANP in children.

In order to evaluate the possible role of vasoactive hormones in the mechanism of exaggerated sodium loss due to reduced renal mass we measured plasma concentration of atrial natriuretic peptide (ANP), aldosterone, plasma renin activity (PRA), plasma noradrenaline, and dopamine, in 12 children with advanced chronic renal failure (mean CIn17.8-2.6,x± SEM, CPAH93.5±17 ml/min per 1.73 m2, FENa7.0±0.95%). No patient had clinical signs of volume overload. Plasma concentrations of ANP were not significantly different from those of healthy agematched controls (29.2±7.2 vs 23.2±3.1 fmol/ml) and did not correlate with urinary sodium excretion. Plasma concentrations of aldosterone, PRA and noradrenaline, were also within the physiological range, while plasma dopamine levels were elevated (260±36 vs 98±11 pg/ml, <0.001). Our data do not support the notion that ANP or the renin-aldosterone axis play a major role in the adaptation of remaining nephrons to maintain long-term sodium balance in normotensive children with chronic renal failure.

The effect of low-dose (2–4 μg/kg/min) and long-term (≥ 46 h) dopamine infusion on serum prolactin and thyrotropin concentrations was investigated in 8 preterm infants with hyaline membrane disease. Dopamine was administered for systemic hypotension and/or for impending renal failure. Serum prolactin decreased from 1,314.5 ± 422.7 μU/ml to 489.9 ± 464.1 μU/ml (p < 0.005), while serum thyrotropin fell from 3.77 ± 2.27 μU/ml to 1.01 ± 0.25 μU/ml (p < 0.025) during dopamine infusion. Our data indicate that exogenous dopamine exerts an inhibitory effect on the secretion of prolactin and thyrotropin even in the sick preterm neonate. The role of prolactin in fetal lung maturation and in regulation of the neonatal tissue water stores is discussed. The results of the present study are also useful in explaining the renal effects of long-term low-dose dopamine infusion in the sick preterm infant.

The role of atrial natriuretic peptide (ANP) in the extracellular volume expansion (ECVE) induced natriuresis was examined in normal man under basal conditions and following dopamine blockade. Hypotonic ECVE was induced by drinking of 20 ml/kg tap water and subsequent intravenous infusion of 2 1 0,9% saline over a period of 4 hours. This maneuver caused an increase in the plasma concentrations of ANP from 25.8 ± 3.4 (x¯+ SEM) to 59.7 ± 6.7 fmol/ml. There was a dissociation between AN¯P response and urinary sodium excretion. A transient rise in glomerular filtration rate (GFR), plasma dopamine and a continuous decrease in plasma renin activity, aldosterone, vasopressin, and noradrenaline were observed. The natriuretic response to ECVE was blunted during dopamine blockade by metoclopramide, but plasma ANP, renin activity, catecholamine and vasopressin levels were not affected. However, plasma aldosterone rose. Our data are compatible with the concept that intrarenal dopamine and raised plasma concentration of ANP contribute to the natriuretic response to ECVE, but these hormonal changes do not completely explain the underlying mechanisms.

Some renal parameters have been studied in newborns with respiratory distress syndrome (RDS). During the first 24 h, the serum creatinine level of the severe cases of RDS are significantly increased, decreasing gradually with improvement of the RDS and reaching normal values within 10 days. Blood urea nitrogen remained unchanged. In the milder cases of RDS, the serum creatinine also showed a slighter increase, which became normal within 4 days after birth. During the acute phase of RDS, there is a reversible impairment in the renal function, which correlates with the degree of hypoxia, hypercapnia and acidosis determined by the primary disease.

The effects of continuous positive airway pressure respiratory therapy – used in centrencephalic apnea in premature infants – has been investigated at a value of + 6 cm H2O end-expiratory pressure. The results showed a significantly decreased glomerular filtration rate, decreased sodium and hydrogen ion excretion and urine output, without changes in systemic blood pressure, pulse rate, and blood gas values. At + 3 cm H2O end-expiratory pressure no changes were observed.

Summary The acute effects of i.v. somatostatin (250 mcg bolus followed by 250 mcg/h continuous infusion for two hours) on renal hemodynamics, renal electrolyte and water handling, and urinary excretion of catecholamines and prostaglandins, as well as on plasma concentrations of arginine vasopressin, atrial natriuretic factor, norepinephrine, epinephrine, dopamine, glucagon, and plasma renin activity were studied in seven normal subjects. Somatostatin decreased effective renal plasma flow and glomerular filtration rate, osmotic and free water clearances, urine volume, and sodium and potassium excretion, while urinary osmolality, fractional excretion of sodium, and phosphate excretion increased significantly. Plasma concentrations of arginine vasopressin, atrial natriuretic factor, norepinephrine, epinephrine, and dopamine remained unchanged, while plasma renin activity (3.0±0.25 vs 2.4±0.2 ng AngI/ml/h;p}<0.01) and glucagon levels (40±11 vs 20±16 pg/ml;p}<0.01) decreased. Urinary excretion of norepinephrine, epinephrine, dopamine, PGE2, and PGF2alpha was suppressed under somatostatin. A significant positive correlation was found between urinary dopamine and sodium excretion (r=0.7;p}<0.001) and urinary postaglandin E2 and glomerular filtration (r=0.52;p}<0.01). Without accompanying changes in plasma osmolality and vasopressin concentration significant antidiuresis occurred, suggesting a direct tubular effect of somatostatin. However, the hormone-induced changes are due mainly to the decrease in renal plasma flow. The results demonstrate that somatostatin at supraphysiological doses exerts significant effects on the kidney.

Abstract: We aimed to study the reproducibility of sodium-lithium countertransport [SLCT] activity and ambulatory blood pressure monitoring [ABPM] in type 1 diabetes. We did this by performing repeated measurements of SLCT activity and ABPM in 11 recent-onset diabetic children and in 11 patients with longer duration of diabetes. Both parameters were related to microalbuminuria. In the older group of diabetic children a significant correlation [r = 0.78; P<0.005] in SLCT activity between the first and second study was observed [514.3±186.4 vs 491.0 ± 148.0 μmol/l erythrocytes/h]. Diurnal systolic and diastolic blood pressure were comparable at both time points within the same group of diabetic children [in group 1: 102.6±6.1 vs 108.6±7.6 mmHg N.S.; in group 2: 113.4 ± 10.6 vs 114.0±7.8 mmHg N.S. Diastolic blood pressure in group 1: 57.4±4.8 vs 65.7±6.9 mmHg N.S., in group 2: 70.6±9.1 vs 68.5±5.3 mmHg N.S.]. Moreover, there was a significant correlation in both diurnal and nocturnal systolic blood pressure between the first and second study in the whole diabetic population. Both SLCT activity and blood pressure values obtained by ABPM were found to be reproducible individual characteristic markers in type 1 diabetic children.

Nine premature infants with birth weight of 1150 to 2500 g and gestational age of 28 to 35 weeks were given dopamine in a dose of 2–4 µg/kg/min to treat cardiopulmonary distress.

Patients with cystic fibrosis (CF) exhibit normal concentrations of sodium and chloride in spite of the disturbance of Cl‐and Na+ transport in epithelial cells. To characterize compensatory mechanisms in the regulation of sodium homeostasis, erythrocytes of 13 CF patients were analysed for sodium‐lithium counter‐transport (SLC), Na+/K+ ‐ATPase activity and intracellular sodium content. Values were compared to those of healthy controls. Patients with CF had normal serum sodium and chloride concentrations and renal excretions of these ions were within the physiological range. Intracellular sodium concentration was similar in the CF and the control group (6.8 ± 2.2 vs 5.7 ± 1.0 mmol/l RBCs). Red blood cells' SLC and Na+/ K+ ‐ATPase activity were elevated in CF patients (381 ± 106 μmol/h/l RBCs vs 281 ± 64; p< 0.01) and (445 ± 129 μmol ATP mg prot/h vs 322 ± 84, p< 0.01). Our study demonstrates that transmembrane cation transport systems are highly activated in CF. The increased sodium transport may be part of a compensatory mechanism of sodium homeostasis in children with CF.

Preeclampsia (PE) is characterised by a systemic maternal inflammatory response and reduced immune tolerance towards the developing fetus. Yet, little is known about the impact of maternal PE on the fetal immune system. We investigated the frequency of distinct immune cell subsets and plasma cortisol and cytokine levels in preterm infants of PE mothers during the first week of life and compared them to preterm infants born from pregnancies not complicated by PE.Cord blood and peripheral blood samples on the 1st, 3rd, and 7th days of life were collected from 14 preterm infants affected by PE (median GA: 30 weeks, BW: 985 g) and 14 controls (median GA: 29 weeks, BW: 1180 g). Ethical approval was obtained from the institution (Semmelweis University). We measured plasma cortisol and cytokine levels and also assessed the prevalence of T, NK and dendritic cell (DC) subsets and activation markers using flow cytometry, using the following antibodies: CD3, CD4, CD8, CD25, CD45RA, CD45RO, CD62L, CD69, CD161, CXCR3, CCR4, HLA-DR, 6 B11, CD11c, CD123.The prevalence of CD4+ T lymphocytes and CD4+HLA-DR + T cells was significantly lower in preterm infants of PE mothers on day 3 compared with controls. The prevalence of memory T cells (CD4+ CD45RO+) was significantly higher in PE infants on day 7. CD8+CD69+ activated T lymphocytes had a lower prevalence on days 0 and 1 in preterm infants born to PE mothers. Myeloid DCs (CD11c+) had a lower prevalence on days 1 and 3 in PE infants. The prevalence of NK cell subsets was comparable in both groups. MCP-1 and IL-4 had significantly higher levels on all 3 postnatal days in infants of PE mothers, while cytokine levels were generally lower at birth compared with controls. Cortisol levels were lower in PE infants on day 1 and 7, respectively.Our observations show that PE has a remarkable impact on the development of the newborn’s immune status during the first week of life by influencing the prevalence of distinct immune cell subsets as well as plasma cytokine and cortisol levels.

The effect of long acting somatostatin analogue, SMS 201-995, on postprandial dumping syndrome was studied in eight patients with Billroth II gastric resection. Each patient was subjected to two oral glucose challenges with 75 g glucose. One challenge was premedicated with 50 μg SMS 201–995 subcutaneously 15 min before the oral intake of glucose, the other with placebo. With placebo all patients experienced the subjective symptoms of the early dumping syndrome with significant (P<0·001) increases (mean (s.d.)) in pulse rate (from 66 (8) to 102 (10) beats/min), in packed cell volume (from 0·36 (0·05) to 0·43 (0·1) l/l) and in the plasma levels of vasoactive intestinal polypeptide (from 3·0 (0·5) to 10·2 (1·8) pmol/l). During the somatostatin study the subjective symptoms and the changes in the various parameters were not detected. In the control study seven patients showed postprandial hypoglycemia. In these patients significant elevations (P<0·001) in the insulin level (from 10 (0·9) to 40 (9·1) μE/ml) and gastric inhibitory peptide (GIP) concentration (from 100 (13) to 220 (41) ng/l) were seen, compared with the initial values. During the application of SMS 201-995 hypoglycaemia did not develop and plasma insulin and GIP concentrations remained unchanged. These results indicate that the long acting somatostatin analogue alleviates the symptoms of early and late postprandial dumping syndromes.

AIM: To study the relation between erythrocyte Na(+),K(+)-ATPase subunit isoform composition, Na(+),K(+)-ATPase activity, and cation pump function in preterm and term neonates. DESIGN: Erythrocyte Na(+),K(+)-ATPase subunit isoform abundance, Na(+),K(+)-ATPase activity, and cation pump function were studied in blood samples obtained from 56 preterm neonates of 28-32 weeks gestation (group 1), 58 preterm neonates of 33-36 weeks gestation (group 2), and 122 term neonates (group 3) during the first two postnatal days. RESULTS: alpha(1) isoform abundance was higher and beta(2) isoform abundance was lower in group 1 than in group 3 (p = 0.0002). alpha(2) and beta(1) isoform abundance did not change with maturation and there was no evidence for the presence of the alpha(3) isoform. Gestational age was inversely related to Na(+), K(+)-ATPase activity (p = 0.0001) and directly related to intracellular Na(+) concentration (p = 0.0025). CONCLUSIONS: Expression of the alpha(1) and beta(2) Na(+),K(+)-ATPase subunit isoforms is developmentally regulated. The increased abundance of alpha(1) isoforms of immature neonates translates to increased ATPase activity. The lower intracellular Na(+) concentration of immature neonates suggests that their erythrocyte Na(+),K(+)-ATPase cation pump function may also be increased.