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Original Articles

Indian Pediatrics 2000;37: 153-158

Free Oxygen radicals in acute renal failure

N.K. Dubey, Praveen Yadav, A.K. Dutta, V. Kumar, G.N. Ray and S. Batra

From the Departments of Pediatric Intensive Care and Biochemistry, Kalawati Sarn Children's Hospital and Lady Hardinge Medical College, New Delhi 110 001, India.

Reprint requests: Dr. N.K. Dubey, A-11/25, Vasant Vihar, New Delhi 110 057, India. E-mail: dubeyn.k@ndfdel.in

Manuscript received: December 17, 1998; Initial review completed: February 2, 1999; Revision accepted: August 30, 1999.

 

Objective: To assess the levels of free oxygen radicals in acute renal failure and their predictive value in clinical outcome. Design: Prospective. Setting: Intensive care unit. Methods: Study was conducted in 50 children (25 with acute renal failue and 25 age and sex matched controls). Blood urea, serum creatinine, serum protein, uric acid and free oxygen radical markers were estimated in both groups. Superoxide dismutase (SOD), glutathione peroxidase(GPx) and lipid peroxide (LPO) wre estimated in blood by standard techniques. Results: Hemolytic uremic syndrome (HUS) was a major cause of acute renal failure (52%), rest were due to acute glomerulonephritis (AGN), septicemia and renal venous thrombosis. In the renal failure group 56% of the patients were dialyzed (peritoneal) and the mortality was 28% (7/25). The levesl of SOD, GPx and LPO were significantly raised in renal failure group. Higher values of LPO, SOD and GPx were documented in subjects who expired. The most important independent variable for predicting clinical outcome was LPO with a sensitivity of 89.4%, specificity of 93%, positive predictive value of 95%. Conclusion: Levels of free oxygen radicals (SOD, LPO and GPx) are raised in acute renal failure and these enzymes can be used as marker of renal injury. LPO levels are highly sensitivity and specific for predicting the clinical outcome.

Key words: Acute renal failure, Free oxygen radicals.

Acute renal failure is a clinical syndrome characterized by rapid decline in glomerular filtration and change in body homeostatic mechanism. It complicates about 5% of the admissions in the hospital and 30% of intensive care unit admissions(1).

Evidence have accumulated over the years incriminating free oxygen radicals as causative agents of renal damage(2_4). The hypoxic ischemia may be less damaging than the large flux of oxygen derived free radicals, which occur during reperfusion of the tissue. Mechanisms involved in generation of the free oxygen radical are injured mitochondria, by xanthine oxidase, by arachidonic acid path-way or by polymorphonuclear cell accumulation in injured tissue(5_8). The renal tubules have high density of mitochondria which shows structural and functional defects in acute renal failure. In addition both xanthine and arachidonic acid metabolism are very active in renal tissue.

Oxygen radicals are capable of reversibly or irreversibly damaging compounds of all biochemical classes including nucleic acid, protein, free aminoacid, lipids, lipoprotein, carbohydrate and connective tissue macro-molecules. These species may have impact on such cell activities as membrane function, metabolism and gene expression. The degree of damage can be assessed by measuring the levels of antioxidants, since they also serve as footprints of oxidant damage(5). The present study was undertaken to evaluate the level of antioxidant enzymes in blood of children with acute renal failure and determine their role in predicting the ultimate outcome of these patients.

Subjects and Methods

Fifty children were recruited from Intensive Care Unit (ICU) and Child Health Promotion Clinic (CHPC) of Kalawati Saran Children's Hospital in two groups of 25 each. Group A (Study) consisted of patients presenting with renal failure who were admitted in ICU and Group B (Control) consisted of age and sex matched normal healthy children attending the CHPC for immunization or nutritional advice. Inclusion criteria for the patients in the study group were: age group between 0-12 years; Acute Renal Failure (ARF), defined as rapid decline in renal function with oligoanuria (<400 ml/m2) and rising levels of blood urea and creatinine with or without acidosis. Electrolyte disturbance and disturbed body fluid (hyper-volemia) were additional criteria. All patients with pre-renal failure who responded to correction of dehydration and obstructive uro-pathy were excluded from the study.

In the study group a thorough clinical examination was done with recording of weight, pulse, temperature, respiratory rate and blood pressure. Patients were given a challenge dose (20ml/kg) of Ringer Lactate if dehydrated. Frusemide or dopamine was used wherever indicated. Blood transfusion was given to patients presenting with signs and symptoms of hemodynamic instability. Majority of these patients were treated with intermittent peritoneal dialysis. Strict input/output charts were maintained with daily weight record. The investigations undertaken in study group were complete hemogram, coagulation profile, peripheral smear for evidence of hemolysis and malarial parasite, X-ray chest and abdomen (KUB region), USG abdomen, urine micro-scopy and culture/sensitivity and arterial blood gas analysis. Blood urea, serum creatinine, serum electrolytes, serum uric acid, serum proteins and free oxygen radical estimation was done in both groups. Three ml heparinized blood was analysed for superoxide dismutase (SOD), glutathione peroxidase (GPx) and lipid peroxide (LPO). Superoxide dismutase was analyzed by method of Mishra and Fridovich(9). One enzyme unit is defined as amount of protein that inhibits the antioxidation of epinephrine by 50% under specified unit. Gluthathione peroxidase was analyzed by method of Leophold and Wolfgans(10) and lipid peroxidase was determined by method of Utley et al.(11).

Statistical significance was assessed by Fischer's test. Multivariate analysis was also used to assess the predictive value of enzymes.

Results

The mean age of children in study group was 4.67±3.89 years compared to 4.83±3.64 years in control group. Six children each in study and control groups were less than or equal to 2 years while in the age group of 2-8 years, there were 13 children in study group and 15 in control. The male: female ratio was equal in both groups.

Table I depicts the etiology of ARF in the study group. Fifty two per cent of the cases were due to hemolytic uremic syndrome, 12% were due to septicemia, 16% due to acute glomerulo-nephritis and in 20% of the patients cause was miscellaneous. Fifty six per cent of the patients were treated with peritoneal dialysis (mean duration 86±34 hours).

A comparison of different biochemical parameters in the two groups is summarized in Table II. Blood urea, serum creatinine and serum uric acid were significantly higher in the renal failure group whereas total proteins and total cholesterol were significantly lower. The evaluated free oxygen radicals (SOD, GPx and LPO) were significantly higher in the renal failure group.

Seven of the 25 patients expired-5 were cases of HUS, 1 of acute glomerulonephritis and one of sepsis. The mean values of urea, creatinine and uric acid were comparable in survivors and those who expired. However the values of LPO, SOD and GPx were signifi-cantly raised (p <0.001) in patients who expired (Table III). The results were further analyzed for their predictive value by using multivariate logistic regression analysis, taking the three enzymes as independent variables and the outcome as dependent variable (survivors - 18 and expired - 7). The analysis revealed that LPO and SOD were both independent predictors of outcome. The other enzyme GPx was not significant in multivariate analysis.

Table IV depicts the predictive ability of various enzymes. The sensitivity of LPO and SOD was high while the specificity was highest for LPO, followed by SOD.

 


Table I__ Etiology and Peritoneal Dialysis Status in Acute Renal Failure
Etiology No. (%) Dialysis
Hemolytic urenic syndrome 13 (52) 9
Sepsis with shock 3 (12) 2
Acute glomerulonephritis  4 (16) 2
Others (Acute tubular necrosis) 5 (20) 1
  25 14

Table II__Comparison of Biochemical Parameters
Parameters Study group (SD)  Control group (SD) `P' value
Blood urea 98.45 (10.67) 24.64 (2.38)  < 0.001
S. Creatinine  2.95 (0.82) 0.35 (0.17) < 0.01
Total proteins 5.40 (1.38) 7.47 (0.86) < 0.001
Total cholesterol 121.43 (19.67) 150.48 (7.38) < 0.01
S. Uric acid 12.35 (1.69) 4.65 (0.69) < 0.001
SOD .830 (5.308) 5.15 (1.98) <0/01
Gap 6.15 (3.350) 1.62 (1.41) < 0.01
LPO  22.85 (7.346) 10.07 (8.68) < 0.001

Table III__Clinical Outcome and Enzyme Levels.
Enzymes Controls (n=25) Survivors (n=18)  Expired (n=17) Cut off values for poor prognosis P value*
SOD 5.150 (1.981)  7.890 (1.937)  10.216 (2.416) 7.983  0.025
GPx 1.618 (1.408) 5.319 (1.633) 8.321 (2.064) 5.846 0.001
LPO 10.08 (8.67) 19.430 (1.437) 31.81 (3.431)  21.328 0.001

Table IV__Comparison of Predictive Ability of Various Enzymes.
Predictive ability (%)  SOD  GPx  LPO  P value*
Sensitivity 3 53.2 89.4 <0.01
Specificity 90 51 93.7 <0.01
Positive Predictive value  74.5 27.5 95.2 <0.01
Negative Predictive Value 71.8 79.1 91.7 <0.1
False negative  18 42 11.1  
False positive 12.1 45 4.5  

Discussion

Almost all tissue components, i.e., lipid, nucleic acid, proteins and carbohydrates are susceptible to free radical induced injury. The most important mechanism of cellular injury is a chain reaction known as lipid per-oxidation(12,13).

The free oxygen radicals (superoxide anion, hydrogen peroxide and hydroxyl radicals) are produced by sequential incomplete reduction of oxygen molecule and are promptly scavenged by antioxidant enzymes present in vivo. Since they are short lived in circulaton it is difficult to directly detect them(3_14). In numerous studies their generation have been assessed indirectly by measuring free oxygen radical mediated lipid peroxidation(4_7). In the present study also, the lipid peroxidation end products and antioxidant enzymes were measured in blood and were found to be significantly raised in subjects with renal failure. Similar results have been reported in uremic patients(15). In this study most of the patients were adolescent and had chronic renal failure. Trznadel et al. have also reported increased concentration of malonyldialdehyde and erythrocyte superoxide dismutase activity in chronic uremic patients undergoing hemo-perfusion and hemo-dialysis(16). Similar observations were made in children with steroid sensitive nephrotic syndrome in whom levels of SOD and LPO were found to be high during relapse(17). Mocon et al. also found high levels of LPO in nephrotic patients in relapse(18).

SOD levels were found to be high in our study suggesting it's role in scavenging the free superoxide radicals. The increased formation of superoxide and hydrogen peroxide radicals produce direct cellular damage. SOD is effective in protecting the reperfusion damage after ischemia in several systems including kidney although it can not protect against damage by ischemia itself. Kontos et al. demonstrated the protective role of superoxide dismutase in birth asphyxia patients receiving SOD exogenously(19). Kohtaro et al. found elevated SOD levels in nucleated RBC of uremic patients(15). Similar observations were made by others(16,20,21).

GPx was also significantly raised in subjects with renal failure. GPx being an antioxidant enzyme removes precursors of free oxygen radicals and is necessary for the conversion of hydrogen peroxide to molecular oxygen and water. However, in another study in nephrotic children, GPx levels were low during relapse(17).

In our study, levels of LPO were significantly higher in 7 patients who expired as compared to survivors (18 patients). The extent of lipid peroxidation (cell membrane damage) denotes the amount of free oxygen radicals generated which have not been scavenged by the defense mechanism. Lipid peroxidation may not be related to the primary tissue injury, but may amplify the original injury. The levels of LPO and SOD correlated well with the severity of disease. Both LPO and SOD were found to be specific and sensitive indicators of clinical outcome in these patients. LPO was highly specific (93.7%) and sensitive (89.4%) whereas SOD was less specific and sensitive. It is possible that the early damage caused by the free oxygen radicals produces significant rise in lipid peroxidation products. The low activity of SOD in plasma and the time lag in activating the scavenging mechanism may be another factor. The inability of the SOD to protect against damage caused by ischemia is also a contributory factor.

In conclusion, levels of free oxygen radicals (SOD, LPO and GPx) are elevated in acute renal failure and the prognostic value of these enzymes requires further confirmation.

References

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16. Trznadel K, Pawicki L, Kedzioraeld T. Superoxide anion generation, erythrocyte super-oxide dismutase activity and lipid peroxidation during hemoperfusion and hemodialysis in chronic uremic patients. Free Radical Bio Med 1990; 8: 201-209.

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children with steroid sensitive nephrotic syn-drome. Pediatr Nephrol 1998; 12: 751-754.

18. Macon H, Aksoy A, Uydu HA, Mocan MC. Oxidative damage of erythrocyte membrane in nephrotic syndrome. Pediatr Nephrol 1999; 13: 326-332.

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