Journal of Human Hypertension (2003) 17, 265–270 & 2003 Nature Publishing Group All rights reserved 0950-9240/03 $25.00 www.nature.com/jhh ORIGINAL ARITICLE Effects of ramipril in nondiabetic nephropathy: improved parameters of oxidatives stress and potential modulation of advanced glycation end products

KSˇ ebekova´1, K Gazdı´kova´1, D Syrova´2, P Blazˇı´cˇek2, R Schinzel3, A Heidland3, V Spustova´1 and R Dzu´ rik 1Institute of Preventive and Clinical Medicine, Bratislava, Slovakia; 2Military Hospital, Bratislava, Slovakia; 3University Wuerzburg, Germany

Enhanced oxidative stress is involved in the progres- patients on conventional therapy did not differ signifi- sion of renal disease. Since angiotensin converting cantly from the ramipril group, except for higher Hcy enzyme inhibitors (ACEI) have been shown to improve levels in the latter. Administration of ramipril resulted in the antioxidative defence, we investigated, in patients a drop in and proteinuria, while creati- with nondiabetic nephropathy, the short-term effect of nine clearance remained the same. The fluorescent the ACEI ramipril on parameters of oxidative stress, AGEs exhibited a mild but significant decline, yet CML such as advanced glycation end products (AGEs), concentration was unchanged. The AOPP and malon- advanced oxidation protein products (AOPPs), homo- dialdehyde concentrations decreased, while a small rise cysteine (Hcy), and lipid peroxidation products. Ramipril in neopterin levels was evident after treatment. The (2.5–5.0 mg/day) was administered to 12 newly diag- mentioned parameters were not affected significantly in nosed patients for 2 months and data compared with a the conventionally treated patients. Evidence that rami- patient group under conventional therapy (diuretic/ pril administration results in a mild decline of fluores- b-blockers) and with age- and sex-matched healthy cent AGEs is herein presented for the first time. The subjects (CTRL). Patients had mild to moderate renal underlying mechanism may be decreased oxidative insufficiency and showed, in the plasma, higher fluor- stress, as indicated by a decline in AOPPs and escent AGE and carboxymethyllysine (CML) levels, as malondialdehyde. well as elevated concentrations of AOPPs, lipofuscin Journal of Human Hypertension (2003) 17, 265–270. and Hcy when compared with CTRL. Basal data of the doi:10.1038/sj.jhh.1001541

Keywords: nondiabetic nephropathy; fluorescent AGEs; carboxymethyllysine; advanced oxidation protein products (AOPPs); malondialdehyde; ramipril

Introduction rise is attributed to enhanced formation, because of carbonyl and oxidative stress.7,8 AGEs exert their Chronic renal insufficiency (CRI) is associated with toxicity both directlyFby alerting the structure/ an enhanced oxidative stress, which is assumed to function of plasma/tissue proteins9Fand indirec- accelerate the progression of renal and cardiovas- F 1,2 tly after their binding to the specific cell surface cular disease. Among the numerous uremic receptorFincluding RAGE.10 This interaction leads toxins, three different compounds show an associa- to the production of reactive oxygen species, which tion with enhanced oxidative stress. (1) Advanced may in turn accelerate formation of AGEs and lipid glycation end products (AGEs) accumulate in the peroxidation products. (2) Homocysteine (Hcy)Fan organism during experimental and clinical human independent risk factor of cardiovascular diseaseF in proportion to the decline in renal 11 3–6 accumulates in circulation of patients with CRI. function. Besides decreased renal removal, their Its auto-oxidation results in the production of reactive oxygen radicals,12 which may contribute to the oxidative stress in the renal disease. (3) Correspondence: Dr K Sˇ ebekova´, Institute of Preventive and Advanced oxidation protein products (AOPPs)F Clinical Medicine, Limbova´ 14, 833 01 Bratislava, Slovakia. F E-mail: [email protected] markers of in vivo protein oxidation accumulate Received 29 May 2002; revised 19 December 2002; accepted 28 in CRI patients consistently, as a consequence December 2002 of impaired renal function.13 They are supposed Ramipril affects AGEs and AOPPs KSˇebekova´ et al

266 to result predominantly from the myeloperoxidase Patients and methods reaction. It still remains questionable whether Patients enhanced oxidative stress may directly potentiate their formation. A high correlation between A total of 12 newly diagnosed individuals with plasma AOPPs and pentosidineFa chemically nondiabetic kidney disease (tubulointerstitial ne- defined AGEFwas revealed.13 phritis, n ¼ 8; glomerulonephritis, n ¼ 2; polycystic Angiotensin-converting enzyme inhibitors (ACEI), kidney disease, n ¼ 2) and impaired renal function alongside their nonhaemodynamic actions, may (serum creatinine 4110 mmol/l or creatinine clear- also improve the defence against oxidative stress. ance o60 ml/min) were administered the ACEI On the one hand, amelioration of the oxidative ramipril (Tritace, Aventis, France) at a dose of 2.5– stress result from the lowered plasma and tissue 5 mg/day over 2 months. Patients’ data were com- angiotensin II (AT II) concentrations. On the other pared to two control groups: (1) seven patients with hand, ACEI containing a sulphydryl moiety in their non-diabetic kidney disease (tubulointerstitial ne- molecule act as free radical and oxidant scavengers, phritis, n ¼ 6; tubulopathy, n ¼ 1) with mild to while lipid peroxidation is inhibited by both moderate renal insufficiency (serum creatinine sulphydryl- and non-sulphydryl-containing ACEI.14 4110 mmol/l or creatinine clearanceo60 ml/min) In in vitro studies, ACEI and AT II type 1 receptor on a long-term antihypertensive treatment with antagonists lower formation of AGEs by inhibition of diuretics and/or b-receptor blockers; and (2) 10 various oxidative steps, as well as the production of age- and sex-matched healthy controls (CTRL), reactive carbonyl species.15 Moreover, in various who were free of any medication 3 months prior to models of experimental renal disease, an attenua- the investigation. To control for the potential tion of oxidative stress following ACEI or AT II seasonal variation in the investigated data: (1) the receptor 1 antagonist (R1A) administration has been patients were recruited into the study within 1 demonstrated.16–19 month, (2) they were instructed not to change their The above-mentioned lines of evidence prompted eating behaviour during the study, (3) blood samples us to investigate whether the protective actions of were taken from patients on conventional therapy ACEI might also be mediated in humans, at least also after the 2-month period for determination of in part, by modulation of the oxidative status. the studied parameters. Group characteristics are Thus, we followed the plasma levels of AGEs, given in Table 1. AOPPs, Hcy and parameters reflecting lipid perox- The study was carried out in accordance with the idation in patients with nondiabetic nephro- Declaration of Helsinki and reviewed and approved pathy during short-term administration of the ACEI by the Institutional Ethics Board. All participants ramipril. signed a written consent.

Table 1 Clinical and biochemical data of the investigated groups of the healthy controls and the patients with nondiabetic nephropathies on the conventional treatment (diuretic/b-blocker) or ramipril before and after 2 months

CTRL n=10 Conv. basal n=7 Conv. 2M Ram basal n=12 Ram 2M

Gender 6F/4M 5F/2M 4F/8M Age (years) 56.0 7 7.5 63.7 7 11.4 62.7 7 13.4 SBP (mm Hg) 136.0 7 11.0 115.0 7 17.1c 116.4 7 15.7c 161.7 7 14.1c 133.8 7 15.7** DBP (mm Hg) 85.5 7 7.6 71.4 7 9.5c 7.52 7 7.0c 97.1 7 4.2c 83.3 7 5.9** Creatinine (mmol/l) 82.7 7 17.6 182.8 7 166.8 175.3 7 168.2 170.9 7 72.7c 168.9 7 68.8c Cl Crea (ml/s) 1.69 7 0.38 0.75 7 0.21c 0.75 7 0.19c 0.72 7 0.52c 0.76 7 0.24c Cystatin C (mg/dl) 0.84 7 0.16 1.42 7 0.85a 1.38 7 0.88a 1.68 7 0.83c 1.65 7 0.80c Glucose (mmol/l) 4.81 7 1.09 4.12 7 0.55 4.54 7 0.72 4.65 7 0.70 4.29 7 0.82 AGE-Fl (AU) 271.0 7 68.1 452.3 7 198.6c 443.7 7 197.1b 455.9 7 137.7c 405.8 7 92.4c,* CML (ng/ml) 528.78 7 229.9 965.8 7 260.4c 1006.0 7 260.4c 888.7 7 456.5c 893.7 7 445.9c AOPPs (mmol/l) 25.96 7 9.85 52.17 7 19.61c 48.28 7 15.7a 53.73 7 21.51c 39.04 7 14.2c,* Neopterin (ng/ml) 1.7 7 0.3 4.3 7 5.3 4.3 7 5.3 4.1 7 1.7c 5.4 7 3.2c MDA (mmol/l) 5.6 7 1.7 5.5 7 2.4 5.0 7 2.3 5.5 7 2.1 3.8 7 1.3**,c Lipofuscin (AU) 24.5 7 2.7 32.3 7 11.6a 31.1 7 13.3 30.1 7 4.2c 30.2 7 3.6c Hcy (mmol/l) 11.7 7 2.6 13.0 7 0.9 13.6 7 1.0 17.6 7 1.4c 18.1 7 1.2c

CTRL: healthy controls; Conv.: conventional treatment; Ram: group treated with ramipril; 2M: values after 2 months treatment; SBP: systolic blood pressure; DBP: diastolic blood pressure; Cl Crea: creatinine clearance; AGE-Fl: fluorescent advanced glycation end products; CML: carboxymethyllysine; AOPPs: advanced oxidation protein products; MDA: malondialdehyde; AU: arbitrary units. aPo0.05 vs CTRL. bPo0.02 vs CTRL. cPo0.01 vs CTRL. *Po0.05 vs 0. **Po0.01 vs 0.

Journal of Human Hypertension Ramipril affects AGEs and AOPPs KSˇebekova´ et al

267 Analytical methods Table 2 Frequency of urinary findings in the investigated groups of the healthy controls and the patients with nondiabetic Venous blood was collected into K2EDTA and nephropathies on the conventional treatment (diuretic/b-blocker) Li-heparin tubes, centrifuged within 1 h after collec- or ramipril before and after 2 months tion and stored at À701C. Plasma was analysed for CTRL Conv. Conv. Ram Ram glucose, creatinine and C-reactive protein (CRP) n=10 basal basal basal 2M concentration employing Vitros 250 analyzer (J&J, n=7 n=7 n=12 n=12 Rochester, USA). Glomerular filtration rate (GFR) was evaluated by determination of plasma cystatin C Proteinuria (>0.3 g/24 h) 0 1 1 4 2 concentration (immunonephelometrically, Dade Microalbuminuria 00154 Behring, Marburg, Germany) and plasma creatinine, (0.15–0.3 g/24 h) Negative ( 0.15 g/24 h) 10 6 5 3 6 with consequent calculation of creatinine clearance. o Analysis of proteinuria was performed by the CTRL: healthy controls; Conv.: conventional treatment; Ram: group pyrogallol red method. treated with ramipril; 2M: values after 2 months treatment. Microalbuminuria was assessed by Micral II test (Boehringer Mannheim, Germany). AGEs were determined fluorimetrically (lex350/lem450 nm, Fluorite 1000, Dynatech, USA)20 and by competitive ELISA (Penzberg, Germany) with the monoclonal antibody 4G-9 directed against Ne-(carboxymethyl)- lysine (CML) as described previously.21,22 Advanced oxidation protein products were determined in plasma spectrophotometrically on a microplate reader (MRX, Dynatech, USA) according to Witko-Sarsat et al,13 malondialdehyde (MDA) levels by HPLC after derivatization with thiobarbituric acid,23 lipofuscin levels turbidimetrically,23 Hcy concentra- tions by employing HPLC,24 and those of plasma neopterin by ELISA (Immunotech, Marseille, France).

Figure 1 Relation between plasma creatinine concentration and Statistics plasma fluorescent advanced glycation end product levels in healthy subjects and newly diagnosed patients with nephropathy. Data are given as mean 7 s.d. For statistical evalua- tion, unpaired Wilcoxon test (CTRL vs patients groups), ANOVA with post hoc least-squares differ- clearance (r ¼À0.689, n ¼ 22, Po0.001). Also, CML ence test (CTRL vs conventional therapy and levels correlated with cystatin C concentrations, rampril) and paired Wilcoxon test (pre- to post- fluorescent AGE levels, as well as with creatinine treatment values) were used. w2 was calculated and a clearance (n ¼ 22, r ¼ 0.664, Po0.001; r ¼ 0.603, regression analysis was performed. 95% confidence Po0.01; and r ¼ 0.844, Po0.001, respectively). All interval (CI) of correlation coefficient was calcu- subjects were normoglycemic (Table 1), and no lated. Po0.05 was considered as significant. relation between AGE-Fl or CML and plasma fasting glucose concentrations was revealed. The mean plasma level of AOPPs was approximately doubled Results in the patients when compared with healthy sub- Healthy controls vs newly diagnosed patients with jects (Table 1). AOPP levels correlated with concen- nephropathy trations of creatinine (Figure 2), cystatin C (r ¼ 0.604, n ¼ 21, Po0.01), and fluorescent AGE levels The patients with nondiabetic nephropathy showed (r ¼ 0.451, n ¼ 21, Po0.05). Lipofuscin levels were a mild to moderate decline of renal function as increased in the patients’ group, while malondial- reflected by serum creatinine, creatinine clearance dehyde levels did not differ between the groups and cystatin C concentrations (Table 1). Data on the (Table 1). Basal fluorescent AGE (Figure 3) and CML occurrence of proteinuria (mean: 1.92 7 0.96 g/24 h) levels (r ¼ 0.645, n ¼ 22, Po0.001) correlated with and microalbuminuria are given in Table 2. Patients lipofuscin if both groups were evaluated together. had mild to moderate hypertension (Table 1) and Moreover, a correlation between AOPP and MDA exhibited higher overall fluorescent AGE levels, as levels was revealed (r ¼ 0.479, n ¼ 21, Po0.05). did the chemically defined AGE, CML. Both AGEs Plasma Hcy concentrations were elevated in the accumulated in circulation in proportion to the patient’s group (Table 1): a mild hyperhomocystei- decline of renal function: fluorescent AGE levels naemia (Hcy 412.5 mmol/l) was observed in 10 correlated positively with serum creatinine (S-Crea, out of the 12 patients with nephropathy, while Figure 1) and cystatin C concentrations (r ¼ 0.648, it was evident in only 1/10 control subjects n ¼ 22, Po0.001), and negatively with creatinine (w2 ¼ 8.983; Po0.01). Hcy concentrations in both

Journal of Human Hypertension Ramipril affects AGEs and AOPPs KSˇebekova´ et al

268 Effect of ramipril administration As expected, ramipril administration resulted in a decline in blood pressure, without affecting renal function (as evaluated by plasma creatinine, creati- nine clearance and cystatin C concentration), during the observed period of 2 months (Table 1). In one out of the four proteinuric patients, proteinuria was normalized, one patient reverted to microalbumi- nuria, and in two a decline was observed (mean: 1.0 7 0.64 g/24 h). In two out of five patients pretreatment microalbuminuria became normal, while in the rest a decline was observed (Table 2). Plasma fluorescent AGE levels decreased slightly Figure 2 Relation between cystain C concentration and plasma but significantly (by 11%), while the intervention advanced oxidation protein product levels in healthy subjects and newly diagnosed patients with nephropathy. did not influence the CML levels. Moreover, under ramipril treatment, the decline in fluorescent AGE levels was accompanied by a decrease in both AOPPs (by 27%) and MDA concentrations (by 31%). Lipofuscin as well as Hcy levels remained unaffected. A trend towards a rise in plasma neopterin levels was revealed (Table 1).

Effect of the conventional treatment After 2 months, no significant changes in the investigated parameters were observed.

Discussion Basal data Figure 3 Relation between plasma lipofuscin and fluorescent advanced glycation end product levels in healthy subjects and Plasma and tissue AGE levels reflect the balance of newly diagnosed patients with nephropathy. their endogenous production26 and their removal. This occurs by scavenging in macrophages,27 as well as by their clearance in the kidney,4–8,28 and liver.29 groups correlated with CML (r ¼ 0.512, n ¼ 22, In our patients, tobacco smoking as a cause of AGE Po0.05) and AGE-Fl (r ¼ 0.432; Po0.05) as well as formation could be excluded, since all participants AOPP levels (r ¼ 0.441, n ¼ 21, Po0.05). Neopterin were nonsmokers. Furthermore, enhanced AGE concentrationsFa marker of monocyte/macrophage formation due to diabetes could be disregarded, as activation25Fwere higher in the patients’ group all subjects were normoglycaemic and reported a when compared with controls. Moreover, there was negative history of diabetes in their parents and a direct relation of neopterin with fluorescent AGE siblings. A microinflammation as a source of (r ¼ 0.571, n ¼ 22, Po0.01), CML (r ¼ 0.681, n ¼ 22, elevation of AGEs seems unlikely, since C-reactive Po0.001), and AOPP levels (r ¼ 0.500, n ¼ 21, protein concentrations were within the normal Po0.05), respectively. range and did not differ between the groups (data not given). Thus, the decline in renal function and Newly diagnosed patients with nephropathy vs the enhanced oxidative stress, as reflected by the conventional treatment group rise in AOPP and lipofuscin levels, could account for the rise of AGE levels. Moreover, elevated Means of the investigated basal parameters of the concentrations of Hcy, which augment the produc- patients assigned to the ramipril treatment group tion of reactive oxygen species,12 could contribute to did not differ significantly from those on long-term AGE accumulation. This assumption is supported conventional treatment, except for higher blood by the correlation between AOPPs as well as Hcy pressure and Hcy values (Table 1). Moreover, and CML levels. Elevated concentrations of neopter- microalbuminuria was negative in all except for in in the patients’ group point to an activation of one patient with mild proteinuria (0.97 g/24 h) monocytes/macrophages as already described in (Table 2). Three patients suffered from mild hyper- early stages of renal impairment.30 The neopterin homocysteinaemia. Its frequency, as evaluated by concentration correlated with both the fluorescent the w2 test, did not differ significantly from that AGE and CML levels, corroborating earlier studies observed in the conventional treatment group. of Witko-Sarsat et al,13 who showed that serum

Journal of Human Hypertension Ramipril affects AGEs and AOPPs KSˇebekova´ et al

269 concentration of the AGE pentosidine correlated with In conclusion, it is suggested that the improved neopterin in patients with impaired renal function. antioxidative defence is involved in the beneficial action of the ACEI ramipril. Effect of ramipril Acknowledgements Treatment with ramipril did not influence the levels of CMLFone of the most prominent AGEs31Fbut This study was supported by a grant from the Slovak resulted in a mild decline of fluorescent AGEs. This Ministry of Health. Support from the Aventis effect could not be attributed to merely seasonal Pharma, s.r.o., Bratislava, Slovakia and the Verein variations, since the comparison of these data Beka¨mzurfung der Hochdruck-und Nierenkrankhei- collected after the 2-month period in patients on ten, Wu¨ rzburg e.V, Germany is acknowledged. We conventional treatment (diuretics and/or b-blockers) thank Professor MUDr. Miroslav Mikulecky´, DrSc. did not show significant changes (Table 1). To the from Institute of Preventive and Clinical Medicine, best of our knowledge there is no prospective data Bratislava for his expert assistance in statistical available on the effect of ACEI or AT II R1A analysis and Mr Andre´ Klassen from Wuerzburg for administration on plasma AGE levels in human an excellent help in preparation of the manuscript. renal disease. However, Forbes et al32 recently demonstrated that rampiril reduced the accumula- References tion of AGEs and attenuated the accumulation of nitrotyrosineFa marker of protein oxidationFin 1 Siems W et al. Oxidative stress in chronic renal failure the model of streptozotocin-induced diabetic ne- as a cardiovascular risk factor. Clin Nephrol 2002; 58 phropathy. Moreover, in our recent long-term study (Suppl 1): S12–S19. (14 weeks) with losartan in subtotally nephrecto- 2 Stenvinkel P et al. 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