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Clinical and Experimental Pharmacology and Physiology (2007) 34, 347–349

doi: 10.1111/j.1440-1681.2007.04552.x

SHORT COMMUNICATION

Blackwell Publishing Asia

ADMINISTRATION OF EXOGENOUS ERYTHROPOIETIN b AFFECTS LIPID PEROXIDATION AND SERUM PARAOXONASE-1 ACTIVITY AND CONCENTRATION IN PREDIALYSIS PATIENTS WITH CHRONIC RENAL DISEASE AND ANAEMIA

Erythropoietin SHORT COMMUNICATION and paraoxonase-1

Judit Marsillach,* Albert Martínez-Vea,† Luis Marcas,† Bharti Mackness,‡ Michael Mackness,‡ Natàlia Ferré,* Jorge Joven* and Jordi Camps* *Centre of Biomedical Research, Sant Joan University Hospital, IRCIS, Reus, †Nephrology Service, Joan XXIII University Hospital, Tarragona, Spain and ‡University Department of Medicine, Manchester Royal Infirmary, Manchester, UK

SUMMARY 1. Patients with advanced chronic renal disease and anaemia have decreased serum paraoxonase-1 (PON1) activity and an increased degree of oxidative stress compared with normal subjects. The present study investigated the effects of treatment of anaemia with exogenous recombinant erythropoietin (EPO) b and iron on levels of antibodies against oxidized low-density lipoproteins (ox-LDL), as well as on serum PON1 activity and concentration, in predialysis patients with chronic renal disease. 2. Forty-nine patients with chronic renal failure and haemoglobin (Hb) < 11 g/dL were treated over a period of 6 months with EPOb (80–120 U/kg per week, s.c.) and variable doses of iron. Selected biochemical variables were determined before and after treatment. 3. Treatment with EPOb and iron was associated with a sig±SD) blood Hb concentration compared nificant increase in mean (± with pretreatment values (12.8 ± 1.5 vs 9.9 ± 0.6 g/dL, respectively; P < 0.001). The average dose of EPOb was 6160 ± 3000 U/week. After 6 months of treatment, compared with pretreatment values, the median levels (95% confidence intervals) of antibodies against ox-LDL were decreased (17.5 (10.6 –24.4) vs 24.8 (11.5– 38.1) U/mL, respectively; P < 0.001), serum PON1 activity was slightly but significantly increased (123.6 (76.1–343.6) vs 101.0 (50.0–332.5) U/L, respectively; P = 0.016) and the concentration of PON1 was significantly decreased (37.3 (11.8 –76.2) vs 46.7 (24.6–98.0) mg/L, respectively; P < 0.001). There were no significant changes in total cholesterol, triglycerides or cholesterol fraction concentrations before and after treatment. 4. We suggest that EPOb and iron treatment of anaemia promotes significant changes in serum PON1 activity and concentration and has a beneficial effect on oxidative stress in predialysis patients with chronic renal disease.

Key words: erythropoietin, lipid peroxidation, paraoxonase-1, renal disease.

INTRODUCTION Cardiovascular complications are the major cause of morbidity and mortality in patients with end-stage renal disease.1,2 According to epidemiological data, the estimated risk for cardiac events is fourto 50-fold higher in advanced renal patients than in the general population.3,4 These patients suffer from alterations in lipoprotein metabolism and composition, chronic volume expansion, anaemia, disturbances in calcium and phosphate metabolism, hyperhomocysteinaemia and a general pro-inflammatory state.5,6 In this respect, oxidative stress has become an interesting emerging issue. Several studies have shown an enhanced production of lipid peroxidation products in advanced renal patients,7–9 together with a low erythrocyte anti-oxidant potential.10,11 Iron treatment, which is used to correct anaemia in these patients, is an additional source of oxidative stress. Paraoxonase-1 (PON1) is an esterase/lactonase that circulates in the plasma associated with high-density lipoproteins and has been postulated to degrade biologically active oxidized lipids and to play a role in an organism’s anti-oxidant system.12 Several studies have reported low serum PON1 activity in patients with end-stage renal disease.13–17 Exogenous erythropoietin (EPO) treatment slows down the progression of renal disease18–20 because it corrects the anaemia and decreases inflammation and oxidative stress in these patients. A recent study showed that EPO administration increased the activity of the anti-inflammatory platelet-activating factor–acetylhydrolase.11 The possibility that long-term EPO treatment influences oxidative stress and serum levels of serum PON1 has not been sufficiently investigated to-date. The present study investigated this in a group of patients with predialysis chronic renal disease and anaemia.

METHODS Correspondence: Dr Jordi Camps, Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, C Sant Joan s/n, 43201 Reus, Catalunya, Spain. Email: [email protected] Received 6 July 2006; revision 14 September 2006; accepted 5 October 2006. © 2007 Blackwell Publishing Asia Pty Ltd

Forty-nine predialysis patients (23 men and 26 women; mean (±SD) age 64 ± 12 years) with chronic kidney disease and a haemoglobin (Hb) concentration < 11 g/dL were treated with EPOb to increase Hb levels to 12–14 g /dL. The cause of chronic kidney disease included chronic glomerulonephritis (eight patients), tubulointerstitial or cystic kidney disease (13 patients),

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vascular nephropathy (12 patients), diabetic nephropathy (11 patients) and was unknown in five patients. Fourteen patients were diabetics and 34 were on angiotensin-converting enzyme inhibitors and /or angiotensin II receptor antagonist treatment. Three patients had stage 3 chronic kidney disease, 24 patients had stage 4 and 22 patients had stage 5. Erythropoietin b (NeoRecormon; Roche Farma, Madrid, Spain) was given at an initial dose of 80–120 U/ kg per week, s.c., one to two times per week. Subsequently, the dose of EPO was adjusted to reach and maintain target Hb levels. Patients received intravenous iron sucrose (Venofer; Uriach-Biohorm, Palau Solità, Barcelona, Spain) or sodium ferric gluconate (Ferrlecit; Watson Pharmaceuticals, Corona, CA, USA) to maintain the transferrin saturation index > 20% and serum ferritin levels > 200 ng/dL. The average doses of EPOb and iron during the study were 6160 U/week (range 1126 –12 000 U/week) and 765 mg (range 0 –2000 mg) over 6 months, respectively. All patients were evaluated at inclusion in the study and after 6 months of treatment using clinical and laboratory assessments. Standard laboratory measurements included Hb, ferritin, creatinine, albumin, calcium, inorganic phosphate, cholesterol, triglycerides and high-density lipoprotein–cholesterol (HDL-C). The concentration of low-density lipoprotein–cholesterol (LDL-C) was estimated using the formula of Friedewald et al.21 Parathyroid hormone was measured by chemiluminiscence (DPC Laboratories, Miami, FL, USA). As a marker of serum oxidative stress, we used the antibody titre against oxidized low-density lipoprotein (ox-LDL), measured by ELISA (Mercodia AB, Uppsala, Sweden). Serum PON1 activity was determined by measuring the rate of hydrolysis of paraoxon at 410 nm and 37°C.22 The serum PON1 concentration was determined by an in-house ELISA.23 The PON1-specific activity was calculated as the ratio between enzyme activity and serum concentration. The normality of distributions was tested using the Kolmogorov–Smirnov test. Differences between measurements before and after EPOb treatment were determined by paired t-test (parametric) or the Wilcoxon rank test (nonparametric). Results are presented as the mean ± SD for Gaussian distributions and as medians (95% confidence intervals) for non-Gaussian distributions. All calculations were performed using spss 13.0 (SPSS, Chicago, IL, USA).

RESULTS Clinical and analytical variables before and after treatment are given in Table 1. As a consequence of EPOb treatment, blood Hb was

significantly increased, whereas there were no significant changes in creatinine clearance, serum cholesterol, triglycerides, ferritin and cholesterol in lipoprotein fractions. After treatment, there was a significant decrease in ox-LDL antibodies, a significant increase in serum PON1 activity and a decrease in serum concentrations of PON1. Therefore, PON1-specific activity was increased significantly. There were no significant relationships between serum PON1 (activity and concentration) and ox-LDL antibodies or renal function. There was a direct significant relationship between levels of ox-LDL antibodies at 6 months and the amount of iron administered (r = 0.35; P < 0.01). We did not observe any significant relationship between PON1 and the aetiology of the disease, the presence of diabetes and treatment with iron or with angiotensin-converting enzyme inhibitors and/or angiotensin II receptor antagonists.

DISCUSSION The results of the present study show that EPOb and iron treatment is associated with a correction of anaemia, decreased levels of ox-LDL antibodies, increased serum PON1 activity and decreased serum PON1 concentration in patients with predialysis chronic renal disease. Interestingly, inhibition of the degree of lipoprotein oxidative stress was achieved despite iron treatment. This was probably related to an improvement in the circulating erythrocyte mass. Previous studies have shown that an improvement of the haematocrit reduces oxidative stress and lipid peroxidation in renal patients by a combination of effects, including an increase in intracellular and plasma levels of anti-oxidants, such as glutathione and superoxide dismutase.9,11,20,23,24 In the present study, an average increase of 30% in the Hb concentration was associated with a similar decrease of ox-LDL antibodies. Although serum albumin is also an efficient antioxidant,24 it could not explain the changes observed in oxidative stress, because the concentration of serum albumin was not changed after EPOb and iron treatment.

Table 1 Characteristics of the chronic kidney disease patients (n = 49) before and after treatment with erythropoietin b Variable BMI (kg/m2) SBP (mmHg) DBP (mmHg) Hb (g/dL) Albumin (g/L) Calcium × phosphate (mmol / L) PTH (pg/mL) Creatinine clearance (mL /min) Ferritin (ng/dL) ox-LDL antibodies (U/mL) Cholesterol (mmol/L) Triglicerides (mmol/L) VLDL-C (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L) PON1 activity (U/L) PON1 concentration (mg / L) PON1-specific activity (U/mg)

Before treatment

After treatment

P value

27.4 ± 4.8 148.3 ± 18.5 80.6 ± 12.2 9.9 ± 0.6 37.8 ± 5.6 3.7 ± 0.8 168.4 ± 122.1 17.7 ± 7.2 236.0 (15.0–1063.1) 24.8 (11.5–38.1) 4.91 ± 1.09 1.44 ± 0.80 0.65 ± 0.36 2.97 ± 0.69 1.28 ± 0.37 101.0 (50.0–332.5) 46.7 (24.6–98.0) 2.45 (0.86–8.38)

27.2 ± 5.0 153.3 ± 24.0 79.4 ± 11.6 12.8 ± 1.5 38.9 ± 4.6 4.3 ± 1.3 178.0 ± 120.7 16.3 ± 8.4 305.7 (25.1–723.2) 17.5 (10.6–24.4) 4.77 ± 1.40 1.39 ± 0.69 0.62 ± 0.31 2.90 ± 0.85 1.28 ± 0.35 123.6 (76.1–343.6) 37.3 (11.8–76.2) 3.81 (1.12–15.39)

NS NS NS < 0.001 NS < 0.001 NS NS NS < 0.001 NS NS NS NS NS 0.016 < 0.001 < 0.001

Results are the mean ± SD, except for ferritin, oxidized low-density lipoprotein (ox-LDL) antibodies and paraoxonase-1 (PON1) which are shown as the median with the 95% confidence interval given in parentheses. BMI, body mass index; SBP, DBP, systolic and diastolic blood pressure, respectively; Hb, haemoglobin; PTH, parathyroid hormone; VLDL-C, very lowdensity lipoprotein–cholesterol; LDL-C, low-density lipoprotein–cholesterol; HDL-C, high-density lipoprotein–cholesterol. © 2007 Blackwell Publishing Asia Pty Ltd

Erythropoietin and paraoxonase-1 Baseline serum PON1 activity and concentration were lower than values observed previously in normal volunteers.25 The relative decrease in activity of the enzyme was higher than that in the protein mass, implying a decrease in PON1-specific activity. (The results in normal volunteers for PON1 activity, PON1 concentration and PON1specific activity were 438.0 (230.8 – 956.2) U/L, 71.4 (50.7–345.2) mg/L and 5.35 (1.04–16.43) U/mg, respectively). Surprisingly, the administration of EPOb was associated with a further decrease in serum PON1 concentration. The reasons for this cannot be ascertained from the present investigation. However, it is known that the PON1 and EPO genes show a genetic linkage together with collagen 1A2 and plasminogen activator inhibitor 1 in a cluster of the long arm of chromosome 7.26 Thus, it is possible that the administration of exogenous EPOb inhibits endogenous EPO expression and, consequently, PON1 expression. The decrease in the serum PON1 concentration was not accompanied by a parallel decrease in serum PON1 activity; in contrast, enzyme activity was increased slightly. This observation is easy to understand in a context of reduced oxidative stress. It is known that lipid peroxides react covalently with a free –SH group at cysteine −284 in PON1, leading to enzyme inactivation.27 Thus, the net result of reduced oxidative stress would be increased PON1 activity. An evident limitation of the present study is the heterogeneous nature of our patients and the differences in treatments. However, we did not observe any significant relationship between these variables and serum PON1 or ox-LDL levels. We suggest that EPOb treatment of anaemia promotes significant changes in serum PON1 activity and concentration and has a beneficial effect on oxidative stress in predialysis patients with chronic renal disease.

ACKNOWLEDGEMENTS This study was supported by grants from the Fondo de Investigación Sanitaria (FIS 05/1607) and the Red de Centros de Metabolismo y Nutrición from the Instituto de Salud Carlos III (C03/08), Madrid, Spain. JM is the recipient of a grant from the Generalitat de Catalunya (FI 05/00068). NF is a researcher from the Juan de la Cierva programme, Ministerio de Educación y Ciencia, Madrid, Spain.

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