Oxidative damage of red blood cells in haemolytic uraemic syndrome

Changes in red blood cell (RBC) lipid peroxidation [measured by malonyl dialdehyde (MDA) concentration], glutathione (GSH) metabolism, antioxidant enzyme activities (catalase, superoxide dismutase, glutathione peroxidase) and haemoglobin (Hb) metabolites (metHb, carboxy Hb) were studied in six children with post-enteropathic (D+) haemolytic uraemic syndrome (HUS) and ten controls. The in vitro effect of hydrogen peroxide [acetyl-phenylhydrazine (APH) test] on GSH and Hb metabolism was also investigated. MDA levels were significantly higher and the antioxidant enzyme activities were lower in HUS patients than in the controls (P<0.01). The oxidised glutathione concentration was significantly higher in the patients than in the control children (26.3±12.6 vs. 10.9±1.8 nmol/g Hb. Percentage values of carboxy Hb and metHb were also higher in HUS (P<0.01). Incubation of RBC with APH induced a more pronounced decrease in the concentration of GSH (P<0.001) and a significant increase (P<0.01) in the level of metHb and carboxy Hb in the HUS patients. This suggests that there is reduced RBC GSH stability in HUS. Utilisation of GSH and antioxidant enzymes leads to increased Hb oxidation and haemolysis. The oxidative damage may have an important role in the pathogenesis of haemolytic anaemia in HUS.     

Acute pancreatitis in six non-transplanted uraemic children

Ten clinical episodes of acute pancreatitis (AP) occurred in six patients (mean age 10 years, range 3–15 years) with chronic renal failure (CRF) during a 9-year period (1977–1986). The underlying cause of CRF was vesicoureteral reflux (2); urethral valves (1); ureterohydronephrosis (1); nephronopthisis (1) and a haemolytic uraemic syndrome which occurred 12 years before (1). In all patients a diagnosis of AP was established both on clinical grounds and with a serum amylase level of >600 IU/1. In 3 patients laparotomy was performed because of suspected appendicitis. All patients required exclusive parentenral feeding (mean duration 25 days) and 2 patients had a partial pancreatectomy. No patient developed pancreatic pseudocysts, 2 patients experienced one relapse (3 and 21 months later) and 1 patient had two relapses and died. Mean duration of follow up was 3 years (range 1–10 years). Possible aetiological factors were: choledochal cyst (1); parotitis without a rise in mumps antibodies (1); familial dyslipidaemia but without AP in other family members (1), and aluminium intoxication with hypercalcaemia and convulsive encephalopathy treated with valproic acid in 1 patient. Severe hyperparathyroidism with radiological signs was absent in all patients. Transplantation had been performed either before AP in 2 patients (1 and 3 years before AP) or had followed AP in 1 patient (7 years after) without occurrence or relapse of AP.This work was presented at the Twenty-First Annual Meeting of European Society for Paediatric Nephrology, 3–5 September 1987, Budapest, Hungary     

A study of recombinant human erythropoietin in the treatment of anaemia of chronic renal failure in children on haemodialysis

This was an open-label multicentre study of recombinant human erythropoietin (r-HuEPO) in 116 children aged 6 months to 20 years with anaemia of chronic renal failure undergoing haemodialysis. Haemoglobin concentration at entry ranged from 3.4 to 9.5 g/dl. r-HuEPO was given intravenously two or three times per week, the starting dose being 75 U/kg per week. This was subsequently titrated in steps of 75 U/kg per week with the goal of increasing haemoglobin concentration at the rate of 1 g/dl per 4 weeks into the range 9.6–11.2 g/dl (6–7 mmol/l), with treatment then continued for up to 1 year with the aim of maintaining the haemoglobin concentration within the target range. Of the 115 children in whom efficacy could be evaluated, 93 (81%) achieved the target haemoglobin and a further 6 had a rise in haemoglobin concentration of at least 2 g/dl. At 52 weeks, the median maintenance dose for children<30 kg was 225 U/kg per week, compared with 107 U/kg per week for children 30 kg. Analysis suggested that 150 U/kg per week would have been a more appropriate starting dose. The mean transfusion requirement fell from 8.9 to 0.7 units/patient per year. Of the 22 patients who failed to reach the target, 15 went on to transplantation and left the study prematurely. Sub-group analysis showed that similar doses lead to similar rates of rise in haemoglobin regardless of the severity of the original anaemia. Assessment of quality of life suggested that this may have improved with r-HuEPO. Twenty-four children needed initiation or increase of anti-hypertensive medication, suggesting that successful r-HuEPO therapy was associated with a tendency towards increased blood pressure. However, there were no significant mean changes in blood pressure, suggesting that the problem was successfully addressed by the changes in treatment. No child developed anti-r-HuEPO antibodies. The overall safety profile was excellent and no new r-HuEPO toxicities were identified in children.     

Erythrocyte susceptibility to oxidative stress in chronic renal failure patients under different substitutive treatments

An increased oxidative stress is now considered one of the major risk factors in chronic renal failure (CRF) patients that may be exacerbated by dialysis. It has been postulated that this increased oxidative stress might cause an augmented red blood cell (RBC) membrane lipid peroxidation with the consequent alteration in membrane deformability. The aim of this study was to evaluate RBC susceptibility to an in vitro induced oxidative stress and RBC antioxidant potential in different groups of CRF patients undergoing different substitutive treatment modalities. Fifteen end-stage CRF patients were evaluated in conservative treatment, 23 hemodialysis (HD) patients, 15 continuous ambulatory peritoneal dialysis (CAPD) patients, 15 kidney transplanted patients, and 16 controls. Their RBCs were incubated with the oxidative stress-inducing agent tert-butylhydroperoxide both in the presence and in the absence of the catalase inhibitor sodium azide, and the level of malondialdehyde (MDA) (a product of lipid peroxidation), was measured at 0, 5, 10, 15, and 30 min of incubation. In addition, the RBC content of reduced glutathione (GSH) was measured by HPLC. As opposed to the controls, RBCs from end-stage CRF patients exhibited an increased sensitivity to oxidative stress induced in vitro, both in the absence and presence of a catalase inhibitor, as demonstrated by a significantly higher level of MDA production at all the incubation times (P < 0.05). Different substitutive treatments had different impacts on this phenomenon; CAPD and kidney transplantation were able to normalize this alteration while HD was not. GSH appeared to be related to the increase in RBC susceptibility to oxidative stress; its content being significantly elevated in end-stage CRF and HD patients as compared with CAPD and transplanted patients and controls (P < 0.05). No significant changes were observed in the RBC glutathione content during the HD session. The increase of GSH in RBCs of end-stage CRF and HD patients seems to indicate the existence of an adaptive mechanism under increased oxidative stress occurring in vivo. Unlike HD, the beneficial effect of CAPD on the anemia of dialysis patients might partly be due to a condition of lower oxidative stress that might in addition counterbalance the cardiovascular negative effects of dislipidemia of CAPD patients.     

Plasma growth hormone-binding activity is low in uraemic children

Plasma growth hormone-binding protein (GH-BP) activity was evaluated in two groups of prepubertal children with chronic renal failure (CRF) who had been treated with recombinant human GH (rhGH). Group 1 consisted of eight children (mean chronological age 10.8 years) with advanced renal failure; group 2 consisted of nine children (mean chronological age 6 years) presenting with end-stage renal disease, who were on dialysis. Before treatment the specific binding of (¹²⁵I)hGH to highaffinity GH-BP was low in the two groups (group 1, 17.3±1.6% of radioactivity; group 2, 14.2±1.4%) compared with the mean value obtained in normal prepubertal children (24.8±1.7%). No significant changes in GH-BP activity were found during the 1st year of GH therapy, although growth velocity and plasma levels of insulin-like growth factor-I increased significantly in both groups. The low GH-binding activity found in children with CRF supports the state of GH resistance. The reason for the absence of a GH-BP response to GH therapy has to be clarified.     

Immunogenicity of hepatitis B vaccine (HEVAC B) in children with advanced renal failure

The immune response after hepatitis B (HB) vaccine HEVAC B was studied in 33 children (mean age 10±4 years) with advanced renal failure. Responders and protected patients were defined by antibody titres to HB surface antigen (anti-HBs) of greater than 10 and 50 mIU/ml, respectively. All received the initial recommended three injections at monthly intervals, and 23 received a booster injection (I_B) 11±1 months after the third injection (I₃). Loss of protection after I₃ led to additional injections in 8 patients (25%). Vaccine was well tolerated and no HB infection occurred during the follow-up period (19±10 months). The percentage of responders was 91% 2±1 months after I₃, and 100% 1 month, 13±1 months and 26±2 months after I_B. The percentages of protected patients at these dates were 91%, 95%, 100% and 100%. Anti-HBs titres 1–3 months after I₃ were useful for indicating those patients likely to have a rapid decline in anti-HBs titres, thus requiring serial anti-HBs determinations and additional injections to prevent a loss of protection. We conclude that, at the expense of a reinforced vaccination schedule in 25% of patients, HEVAC B vaccine can safely achieve a sustained protection in more than 90% of uracmic children.     

Glycation free adduct accumulation in renal disease: the new AGE

Glycation adducts formed in the later stages of protein glycation reactions, advanced glycation endproducts (AGEs), are a class of uraemic toxin. Protein glycation was viewed originally as a post-translational modification that accumulated mostly on extracellular proteins. We now know that AGE residues are also formed on short-lived cellular and extracellular proteins. Cellular proteolysis forms AGE free adducts from these proteins, which are released into plasma for urinary excretion. AGE free adducts are also absorbed from food. AGE free adducts are the major molecular form by which AGEs are excreted in urine. They normally have high renal clearance, but this declines markedly in chronic renal failure patients, leading to profound increases in plasma AGE free adducts. Accumulation of plasma AGE free adducts is increased further in end stage renal disease patients on peritoneal dialysis and haemodialysis by increased AGE formation. The impact of AGEs absorbed from food is probably most marked for undialysed patients with mild uraemia. The toxicity of AGEs has been associated with resistance of the extracellular matrix to proteolysis and AGE receptor-mediated responses. AGE free adducts may also contribute to vascular disease in uraemia. They represent an important new age for glycation research in nephrology.     

Mathematical modelling of haemodialysis in children

The single-pool urea kinetic model (UKM), utilising Kt/V (the normalised whole body urea clearance), is widely used to help assess the adequacy of haemodialysis in adults. In the presence of an adequate dietary protein intake, a value of unity is acceptable for thrice weekly dialysis. Children could benefit from this approach but, with their relatively higher protein intakes and dialysis needs, this model may not be applicable. Urea kinetics, studied in six children with chronic renal failure by serial timed blood urea measurements during and after haemodialysis, were compared with the kinetics of a one-pool and a two-pool UKM. The two-pool UKM with intra- and extracellular pools best fitted the observed data, re-equilibration between pools accounting for the marked rebound increase in blood urea seen in the 1 st h after dialysis ( 17%, SD 5). Kt/V calculated using the end-dialysis blood urea was higher ( 21%, SD 5) than when the more correct equilibrated value was used. The post-dialysis rebound indicates significant disequilibrium between the two pools at the end of dialysis. Dialysis efficiency may be substantially overestimated unless this is allowed for by using the rebounded post-dialysis blood urea when calculating Kt/V.     

Psychosocial issues in children on hospital haemodialysis: nurses' views

In a sample of 21 children and adolescents on hospital haemodialysis, nurses identified a substantial percentage of the children (about half) as having marked problems in psychological adjustment. Nearly half of the children were also regarded as showing poor compliance with aspects of treatment other than dialysis. More children were rated as disturbed by nursing than by research psychiatric assessments; there was limited congruence between nurses' and parent/child ratings of problems in physical well-being or in treatment compliance. The reasons for these discrepancies are explored. Our results highlight the importance that psychological aspects in the nursing of children with chornic renal failure on hospital haemodialysis are recognised.     

Low hair selenium and plasma glutathione peroxidase in children with chronic renal failure

Selenium (Se) is a trace element that incorporates into the selenoenzyme glutathione peroxidase (GSH-Px). There are conflicting results regarding the Se levels and activity of GSH-Px in adult uremic patients. The aim of this study was to determine (1) the hair Se status, (2) the possible relation between the hair Se status and the antioxidant enzyme, GSH-Px, and (3) the influence of different treatment procedures on hair Se status and GSH-Px activity in children with CRI, those treated conservatively and those on HD and on CAPD. Ninety-three patients, including 32 patients with CRI, treated conservatively, 42 PD patients, 19 HD patients and 34 healthy children were enrolled in the study. The hair Se level was measured by the atomic absorption spectrophotometer method. Plasma GSH-Px activity was determined using a Randox test combination (RANSEL). Hair Se levels were significantly lower in the CRI, CAPD, and HD groups when compared to the control group (P=0.001, P=0.001, and P=0.001, respectively). Plasma GSH-Px activity was significantly lower in the CRI, CAPD, and HD groups when compared to the control group (P=0.001, P=0.001, and P=0.001, respectively). Plasma GSH-Px activity correlated with the GFR in patients with CRI and the control group (P=0.000; r²=0.60). There was no correlation between plasma GSH-Px and hair Se levels in the patient and control groups. These results revealed a decreased hair Se level and impaired antioxidative capacity in children with CRI on CAPD and HD. The lack of any relation between plasma GSH-Px and hair Se suggests that plasma GSH-Px is not a good marker of Se stores.     

The role oxygen free radicals and prostaglandins in reperfusion injury to warm ischemic kidneys

Summary The present study was designed to determine whether the administration of superoxide dismutase (SOD) can alleviate ischemic kidney damage and whether there is a relationship between oxygen free radicals and thromboxane (Tx). In 17 dogs, the right kidney was removed and the vascular pedicle of the left kidney was clamped for 75 min. Prior to reperfusion, the ischemic kidney was rinsed with 5 mg SOD and an additional 20 mg SOD was infused systemically. Blood samples were drawn from the renal vein before ischemia and after reperfusion to determine serum levels of thromboxane B₂ (TxB₂). All eight untreated dogs died within 1 week of renal failure, and the nine treated dogs demonstrated transient renal failure, with a significant difference (P<0.001) being found between the two groups. A significant difference (P<0.001) in TxB₂ levels was found in the untreated dogs before and after ischemia and beween the two groups following reperfusion. Animals that are treated with SOD after the ischemic event has occured but before reperfusion exhibit a favorable clinical course in terms of survival and renal function. Tx synthesis in the kidney can be blocked by the administration of SOD.     

Lipid peroxidation and antioxidant enzymes in children with chronic renal failure

Increased lipid peroxidation (LP) has been observed in dialysis patients and in predialysis adults with advanced chronic renal failure (CRF). The aim of this study was to investigate whether predialysis CRF children have increased LP in plasma and red blood cells (RBC) and to evaluate the activity of the antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)] in RBC. Concentrations of selenium (Se), copper (Cu), and zinc (Zn)—cofactors of these enzymes—were determined both in erythrocytes and in plasma. LP was monitored by plasma and erythrocyte malonyldialdehyde (MDA) and by plasma organic hydroperoxide (OHP) concentrations. Forty-six predialysis children, aged 5–18 years, divided into two groups according to their serum creatinine levels [group I (n=14, mean serum creatinine 421.61±141.08 mol/l), group II (n=32, mean serum creatinine 174.94±45.50 mol/l)] and 27 age-matched healthy subjects were enrolled in the study. Significantly higher concentrations of plasma and erythrocyte MDA and plasma OHP, significantly lower activities of GSH-Px and CAT, and significantly lower concentrations of erythrocyte Se, Cu, and Zn and plasma Se and Cu were found in both groups of renal patients compared with controls. The SOD activity was reduced in both groups of CRF children. In group I the activity of SOD and GSH-Px was significantly lower than in group II. In summary, there is increased LP in plasma and RBC in children with predialysis CRF, even those patients with moderate renal insufficiency. The activity of the enzymatic antioxidant defense system is reduced in the RBC of predialysis patients. The antioxidant capacity is related to the severity of renal failure.     

Paraoxonase, anti-oxidant response and oxidative stress in children with chronic renal failure

Increased oxidative stress is believed to contribute to an increased risk of cardiovascular disease in uraemia. In children with chronic renal failure (CRF), an anti-oxidant enzyme, paraoxonase (PON), that inhibits oxidation of LDL-cholesterol, has not been previously investigated. In this study we aimed to investigate PON activity, total anti-oxidant response (TAR), total peroxide (TPX), oxidative stress index (OSI) and some pro-oxidant cytokines in 29 children with CRF [mean age 10.2±3.5 years; 19 pre-dialysis, ten on continuous ambulatory peritoneal dialysis (CAPD)] and in 25 control subjects. Children with CRF had lower PON and TAR and higher TPX and OSI values than did controls (P<0.05). Except for lower TAR and serum albumin levels of the CAPD subgroup (P<0.05), other parameters were similar in non-dialysis and CAPD patients (P>0.05). Patients had significant positive correlation between TAR and serum albumin (P<0.05). Serum urea had significant positive correlation with TPX and OSI (P<0.05). Increased oxidative stress and decreased anti-oxidants measured by serum PON activity and TAR were found in children with CRF. We can hypothesize, on the basis of statistical correlations, that low levels of serum albumin and high levels of uraemic metabolites might be responsible for increased oxidative stress in children with CRF. Further studies with larger sample sizes are needed to verify these results.     

Oxidative stress, inflammation and early cardiovascular damage in children with chronic renal failure

The relationship between inflammation, oxidant stress and cardiovascular damage in children with chronic renal failure (CRF) has not previously been investigated. The aim of this study was to investigate markers of oxidative stress, inflammation and early cardiovascular abnormalities. Therefore, erythrocyte superoxide dismutase (SOD) and catalase (CAT) activities; blood glutathione (GSH) and serum malondialdehyde (MDA) levels; C-reactive protein (CRP) and proinflammatory cytokines (IL-6, TNF-α,); and left ventricular masses (LVM) and intima media thicknesses (IMT) were measured in children with CRF. A total of 29 children with CRF (19 nondialysis, 10 peritoneal dialysis) were included. The control group consisted of 25 healthy subjects. CRF children had significantly increased IL-6, TNF-α, CRP and MDA concentrations and decreased SOD, CAT and GSH levels compared with controls (P<0.05). Nondialysis and peritoneal dialysis subgroups had similar oxidative stress and inflammation biomarkers (P>0.05). Erythrocyte CAT was positively correlated with CRP, TNF-α, and IL2-R in the study group. Positive correlations were found between cytokine concentrations, CRP and urea/creatinine levels. Significantly increased LVM and IMT values were found in CRF children (P<0.05). In conclusion, increased oxidant stress and inflammation together with early cardiovascular damage were found in CRF children. Further studies with more patients are needed to verify these results.     

Plasma levels of advanced glycation end products in children with renal disease

In adults, advanced glycation end products (AGEs) rise slowly in tissues and circulation during aging, and accumulate at an accelerated rate both in diabetes and chronic renal insufficiency (CRI). We aimed to investigate the pattern of AGE accumulation in children/adolescents with CRI and on renal replacement therapy by dialysis and transplantation. Concentrations of fluorescent AGEs, carboxymethyllysine (CML) and lipofuscin-like substance (LFLS, a marker of lipid peroxidation) were followed. Data were obtained from 11 CRI patients on conservative treatment (age 12.6±1.7 years, serum creatinine: 205.7±17.5 μmol/l), ten patients on renal replacement therapy with dialysis (13.6±1.7 years, 698.2±48.9 μmol/l) and nine patients after kidney transplantation (15.9±1.1 years, 115.9±12.0 μmol/l) and comparison made with the data from 28 healthy controls (11.8±8.2 years, 44.1±8.2 μmol/l). In controls, an age-dependent rise of fluorescent AGE and CML levels was observed. In the CRI group, fluorescent AGEs [0.38±0.03×10⁵ arbitrary units (AU)] and CML (369±26 ng/ml) concentrations were doubled compared with controls (0.16±0.03×10⁵ AU and 189±42 ng/ml, respectively) and even higher levels were revealed in dialyzed patients (0.80±0.05×10⁵ AU; 650±94 ng/ml). Successful kidney transplantation significantly reduced but did not normalize fluorescent AGE levels (0.39±0.03 ×10⁵ AU), while the decline in CML levels (550±47 ng/ml) was insignificant. Plasma LFLS was elevated in CRI (19.6± 1.7 AU) and was even higher in dialyzed children (32.0±5.3 AU) compared with healthy controls (7.1± 1.4 AU). Kidney transplantation did not normalize LFLS levels (20.3±5.3 AU), pointing to persistently enhanced lipid peroxidation. Our study provides the first data on enhanced fluorescent AGEs and CML levels in children/adolescents with CRI and on dialysis. Successful renal transplantation decreased but did not normalize AGE levels, probably because of still-impaired renal function with enhanced oxidative stress, as well as the influence of immunosuppressive therapy. Received: 13 July 2000 / Revised: 8 June 2001 / Accepted: 12 July 2001