Endogenous nitric oxide synthase inhibitors and renal ... - CiteSeerX

European Journal of Clinical Investigation (2003) 33, 370–375

Endogenous nitric oxide synthase inhibitors and renal perfusion in patients with heart failure Blackwell Publishing Ltd.

J. T. Kielstein†, S. M. Bode-Böger*, G. Klein†, S. Graf, H. Haller† and D. Fliser† *

University, Magdeburg, Magdeburg, †Medical School Hannover, Germany

Abstract

Background Patients with heart failure are characterized by impaired nitric oxide-dependent endothelial vasodilation and, in addition, by reduced renal perfusion. Design We assessed blood concentrations of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) as well as renal haemodynamics to compare normotensive patients with mild heart failure (n = 12, seven males, 70 ± 1 years, 72·0 ± 2·7 kg, 92 ± 2 mmHg, NYHA I/II) and healthy subjects matched with respect to gender, age and body weight (n = 12, seven males, 69 ± 2 years, 72·7 ± 2·5 kg, 88 ± 2 mmHg). Results Plasma ADMA concentration and renovascular resistance (RVR) were significantly higher (P < 0·01) and effective renal plasma flow (ERPF) significantly lower (P < 0·01) in the patients with heart failure (ADMA 4·18 ± 0·42 µmol L−1, RVR 159 ± 12 mmHg mL−1 min−1, ERPF 381 ± 26 mL min−1 1·73 m−2) as compared with the healthy controls (ADMA 2·38 ± 0·11 µmol L−1, RVR 117 ± 8 mmHg mL−1 min−1, ERPF 496 ± 19 mL min−1 1·73 m−2). In contrast, plasma concentrations of -arginine, homocysteine, symmetric dimethylarginine (i.e. the biologically inactive stereoisomer of ADMA) and plasma renin activity were not significantly different in both groups studied. In the multiple regression analysis, only plasma ADMA concentrations independently predicted reduced ERPF (r = −0·57; P < 0·003). Conclusions In normotensive patients with heart failure plasma ADMA concentrations are markedly increased and related to reduced renal perfusion. Thus accumulation of this endogenous nitric oxide inhibitor may play a role in renal pathology in these patients. Keywords Asymmetric dimethylarginine, effective renal plasma flow, heart failure, renovascular resistance. Eur J Clin Invest 2003; 33 (5): 370–375

Introduction Reduced nitric oxide (NO)-dependent vasodilation has been documented in patients with heart failure [1,2]. Nitric oxide synthase (NOS) synthesizes NO from the amino acid

Division of Nephrology Medical School Hannover, Germany (J. T. Kielstein, S. Graf, H. Haller, D. Fliser); Institute of Clinical Pharmacology, Otto-von-Guericke University, Magdeburg, Germany (S. M. Bode-Böger); Department of Cardiology, Medical School Hannover, Germany (G. Klein). Correspondence to: Jan T. Kielstein, Division of Nephrology, Department of Internal Medical, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Tel.: +49–511–532 6319; fax: +49–511–55 23 66; e-mail: [email protected] Received 22 November 2002; accepted 6 January 2003 © 2003 Blackwell Publishing Ltd

-arginine. Guanidino-substituted analogues of -arginine such as asymmetric dimethylarginine (ADMA) can selectively inhibit NOS by competitive blockade of its active site [3,4]. Several recent clinical studies examining different populations revealed that increased plasma ADMA levels are associated with cardiovascular morbidity and can even predict increased cardiovascular mortality [5–11]. Thus this endogenous NOS inhibitor has emerged as a novel cardiovascular risk factor [12]. We have recently documented that plasma ADMA concentrations markedly increase with senescence, particularly in the elderly with hypertension [13]. In addition, a close relationship between high ADMA blood levels and increased renovascular resistance was observed in these patients. This finding is compatible with the notion that accumulation of ADMA may be responsible for the reduction of renal perfusion, because it is known that (postglomerular) renal circulation is particularly sensitive to NO inhibition [14 – 16]. Observation in laboratory animals suggest that plasma

ADMA and renal perfusion in heart failure

ADMA levels are increased in experimental heart failure [17], but to date no specific information on plasma ADMA levels and their relationship with renal haemodynamics in patients with heart failure is available. In order to address this issue we assessed renal haemodynamics and blood concentrations of -arginine and dimethylarginines in normotensive patients with mild heart failure and in healthy subjects matched with respect to age, gender and body weight.

Methods Participants and protocol The study protocol was approved by the local Ethics Committee; all participants gave informed consent. Twelve patients (seven males, mean age 70 ± 1 years, mean body weight 72·0 ± 2·7 kg), who had undergone heart catheterization and in whom mild heart failure (NYHA I /II) was newly diagnosed, were included into the study. Nine patients had coronary heart disease and three had valvular disease. All patients were compensated at the time of the study, and in none of them was oedema, S3 gallop or jugular venous distension present on physical examination. With the exception of coronary heart disease, and valvular heart disease, none of the patients had further relevant medical problems. In addition, 12 healthy subjects matched with respect to age, gender and body weight were examined (seven males, mean age 69 ± 2 years, mean body weight 72·7 ± 2·5 kg). Manifest atherosclerotic vascular disease and/or heart failure were excluded by clinical examination and echocardiography. To exclude individuals with primary renal disease sonography, urine analysis (including phasecontrast microscopy), and serum chemistry were performed in all participants. Only nonsmoking subjects with normal plasma creatinine concentration were studied. In none of the participants was anaemia present. All participants were studied under outpatient conditions. Dietary counselling was given to all participants, who were advised to ingest a standardized diet with respect to sodium chloride (100 mmol day−1) and calorie content (30 kcal kg−1 body weight) 1 week before and during the examination. All patients with heart failure were studied before an ACE inhibitor therapy was started. All other cardiovascular drugs with the potential to confound the measurement of renal haemodynamics were discontinued in patients before the examination in accordance with their respective pharmacodynamic half-life (with exception of β-blockers). True glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured after 12 h of fasting in a quiet room and supine position using the inulin (Cin) and paraaminohippurate (CPAH) infusion clearance techniques, as described earlier [18]. In brief, a priming dose of 1500 mg of inulin m−2 (InutestR, Laevosan Co., Linz Austria) and 500 mg of paraaminohippurate m−2 (Nephrotest®, BGA, Lich, Germany) was followed by a continuous infusion of inulin (10 mg m−2 min−1) and paraaminohippurate (8 mg m−2 min−1) with ultraprecise pumps (Perfusor FTR,

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Braun, Melsungen, Germany). After an equilibration period of 100 min, blood samples for determination of Cin and CPAH were taken at regular intervals. In order to calculate renovascular resistance, mean arterial blood pressure (MAP) was measured at the same time points during the clearance studies using a noninvasive oscillometric technique (Dinamap®, Criticon Inc., Tampa, FL, USA). Further, blood samples for measurement of methylarginines, arginine, total homocysteine and cholesterol concentrations and plasma renin activity (PRA) were taken without venous compression at the start of the clearance measurement after at least 100 min of being in supine position. In addition, ambulatory 24-h blood pressure was assessed on a separate day using an automatic blood pressure monitoring device (model 90207R, SpaceLabs Inc., Issaquah, WA, USA).

Measurements and calculations Inulin was measured enzymatically using inulinase and paraaminohippurate photometrically. The clearances of inulin and PAH were calculated from the delivered dose: C = (Ir × Ic ) /Sc; where C is the clearance, Ir is the infusion rate (ml min−1), Ic is the concentration of the analyte in the infusion fluid (mg mL−1), and Sc is the plasma concentration of the analyte (mg mL−1). Filtration fraction (FF) was calculated as the ratio Cin /CPAH and renovascular resistance (RVR) was calculated using the equation: RVR = [(MAP – 12) × 723/ERPF]. Plasma -arginine and methylarginine levels were determined by high performance liquid chromatography (HPLC) using precolumn derivatization with o-phthalaldehyde (OPA), as described previously [19]. Plasma samples and internal standards were extracted on CBA solid-phase extraction cartridges (Varian, Harbor City, CA, USA). The eluates were dried over nitrogen and dissolved in bi-distilled water for HPLC analysis. Samples and standards were incubated for 30 s with the OPA reagent (5·4 mg mL−1 OPA in borate buffer, pH 8·5 containing 0·4% mercaptoethanol) before automatic injection into the HPLC. The OPA-derivatives of -arginine, ADMA and symmetric dimethylarginine (SDMA) were separated on a C6H5 column (Macherey and Nagel, Düren, Germany) with the fluorescence monitor set at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. Symmetric dimethylarginine is the biologically inactive sterioisomer of ADMA. The coefficients of variation of this method are 5·2% within assay and 5·5% between assay; the detection limit of the assay is 0·1 µmol L−1. Plasma total homocysteine (Hcy) concentrations were measured with a fluorescence-polarization immunoassay, and PRA with a radioimmunoassay (normal range for supine position: 0·3– 0·6 ng AI mL−1 h−1). All other measurements were carried out with routine laboratory tests using certified assay methods.

Statistics The SPSS package was used for statistical analysis. Normality of data distribution was confirmed with the

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Shapiro-Wilk test. Data of both groups were compared using a t-test for random data. Multiple stepwise regression analysis was performed between ERPF as the dependent variable and age, body weight, MAP, ADMA, SDMA, -arginine, Hcy, cholesterol and PRA as independent variables. The same analysis was repeated with RVR as the dependent variable. The zero hypothesis was rejected at a P-level 0·05. All data are presented as mean ± SEM.

Results Heart failure patients were well matched to healthy subjects with respect to gender, age and body weight. The differences between both groups concerning these variables were not significant. In addition, average 24-h ambulatory MAP was within the normal range in the patients with heart failure and in the normotensive controls; neither was the difference between the groups significant (Table 1). Further, GFR was modestly but not significantly lower in the patients as compared with the healthy subjects, whereas ERPF was significantly lower in the patients with heart failure (Table 1). Conversely, RVR and FF were markedly higher in the group of patients as compared with the healthy individuals. Asymmetric dimethylarginine blood concentrations were significantly higher in the patients with heart failure than in the healthy subjects. Individual data on plasma ADMA concentrations are shown in Fig. 1. In contrast, blood SDMA, -arginine, Hcy and cholesterol levels were comparable in both groups studied (Table 1). In addition, PRA was not significantly different in the patients with mild cardiac dysfunction and the healthy controls. The results of the multiple stepwise regression analysis for determinants of ERPF and RVR are summarized in Table 2. After adjustment for potential confounding by age, body weight, MAP and blood SDMA, -arginine, Hcy and

Figure 1 Individual values of plasma asymmetric dimethylarginine concentration (ADMA) in healthy subjects (mean age 69 ± 7 years) and patients with heart failure (mean age 70 ± 5 years). Symbols: closed symbols = men; open symbols = women; — = mean value.

cholesterol levels and PRA, only plasma ADMA concentration were independently related to ERPF. In addition, the level of blood pressure and ADMA predicted the increase of RVR. The relationship between blood pressure and RVR is not unexpected, however, because MAP is used for calculation of RVR. Thus i MAP is removed from the regression analysis, ADMA (r = 0·42; P < 0·044) remains the only determinant of RVR.

Discussion The results of the present study document that markedly increased plasma concentrations of the endogenous NOS inhibitor ADMA are present even in nonsmoking normotensive patients with mild heart failure. Moreover, plasma

Table 1 Indices of renal function, blood pressure and metabolic variables in the study populations

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Serum creatinine (mg dL ) GFR (mL min −1 1·73m−2) ERPF (mL min −1 1·73m−2) Filtration fraction (Cin CPAH−1) RVR (mmHg mL−1 min−1) 24 h MAP (mmHg) Plasma renin activity (ng AI mL−1 h−1) Plasma ADMA (µmol L−1) Plasma SDMA (µmol L−1) Plasma -arginine (µmol L−1) Plasma homocysteine (µmol L−1) Serum cholesterol (mg dL−1)

Patients with heart failure

Healthy subjects

(n = 12)

(n = 12)

0·99 ± 0·04 92 ± 4 381 ± 26 0·25 ± 0·01 159 ± 12 92 ± 2 0·37 ± 0·06 4·18 ± 0·42 0·53 ± 0·03 58·0 ± 2·7 11·5 ± 0·7 206 ± 8

0·95 ± 0·04 100 ± 2 496 ± 19* 0·20 ± 0·01* 117 ± 8* 88 ± 2 0·41 ± 0·03 2·38 ± 0·11* 0·51 ± 0·03 55·3 ± 1·7 10·4 ± 0·9 203 ± 8

24-h MAP = 24-h mean arterial blood pressure (by Spacelab). GFR = glomerular filtration rate; ERPF = effective renal plasma flow; RVR = renal vascular resistance; ADMA = asymmetric dimethylarginine. * P < 0·01: patients with heart failure vs. healthy controls. © 2003 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 33, 370 –375


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Table 2 Multiple regression analysis for determinants of effective renal plasma flow and renovascular resistance Effective renal plasma flow

ADMA MAP Hcy Age SDMA Chol BW L-Arg PRA

Renovascular resistance

coefficient

P

−0·57 −0·22 −0·19 −0·16 −0·14 −0·11 0·10 −0·09 0·07

0·003 0·22 0·30 0·39 0·45 0·56 0·59 0·63 0·71

MAP ADMA SDMA Age Chol L-Arg BW Hcy PRA

coefficient

P

0·52 0·45 0·28 0·21 0·21 −0·07 −0·04 0·03 −0·01

0·004 0·012 0·10 0·20 0·22 0·68 0·84 0·85 0·92

MAP = mean arterial blood pressure; BW = body weight; ADMA = asymmetric dimethylarginine; SDMA = symmetric dimethylarginine; L-Arg = -arginine; Hcy = homocysteine; Chol = cholesterol; PRA = plasma renin activity.

ADMA levels indepedently predicted the decrease of effective renal plasma flow and increase of renovascular resistance in these patients. In contrast, this was not the case with -arginine, i.e. the substrate for NOS, nor with SDMA, i.e. the stereoisomer of ADMA, which has no inhibitory effect on NOS. Thus despite the relatively small number of thoroughly matched patients studied, our results indicate that increased blood concentrations of the endogenous NOS inhibitor ADMA are linked to reduced renal perfusion in patients with heart failure. This finding is of particular interest, because several recent large studies have revealed that even a minor impairment of renal function predicts overall mortality in patients with cardiovascular disease, and particularly in patients with heart failure [20,21]. Our findings are of interest with respect to the pathophysiology of impaired renal function in patients with heart failure. Theoretically reduced systemic blood pressure and, as a consequence, reduced perfusion of the kidney is thought to be responsible for reduction of GFR and the even more pronounced decrease of ERPF and an increase of renovascular resistance [22,23]. Our results support the notion that reduced availability of NO as a result of NOS inhibition may be involved in this process as well. In this context it has to be pointed out that (postglomerular) renal microvasculature is particularly sensitive to NOS inhibition [14 –16]. This has also been recently demonstrated by using ADMA in animal experiments, in studies with isolated organs, and in studies in healthy volunteers [24,25]. With infusion of ADMA doses that resulted in plasma levels encountered in patients with cardiovascular disease we observed a significant reduction of cyclic GMP, i.e. the main second messenger of NO in the cardiovascular system. In parallel, ADMA infusion caused a significant decrease of ERPF and increase of RVR [26]. Increased ADMA blood levels in patients with heart failure may therefore reduce the availability of NO within the kidney, and thus contribute to endothelial dysfunction and finally lead to increased renovascular resistance. According to several studies in different populations even a small increase in mean plasma ADMA levels is associated with deterioration of endothelial function and a significant increase in the rate of cardiovascular events [4 –11].

Recently published results of a small study suggest that treatment with ACE-inhibitors and angiotensin II receptor blockers in patients with essential hypertension may reduce ADMA blood levels, but the mechanisms of this action were not clarified [27]. We examined our patients with heart failure before initiation of an ACE inhibitor therapy in order to exclude the confounding influence of this therapy on measurements of renal function. Furthermore, it is known that severe heart failure activates neuro-humoral systems such as the renin-angiotensin and sympathetic system [28,29]. For this reason we have examined only patients with mild cardiac dysfunction (and normal serum creatinine concentration) in order to minimize the effects of increased activity of neuro-humoral systems, particularly the reninangiotensin system, on renal haemodynamics. Mean PRA measured in supine position was within the normal range in both groups examined and comparable in the patients with heart failure and the healthy controls. This finding is in line with results of large controlled clinical trials in which no significant activation of the renin-angiotensin system was observed in patients with only mild cardiac dysfunction and without manifest congestive heart failure [28,29]. Thus our results suggest that at least in patients with mild heart failure, increased activity of the renin-angiotensin system does not contribute to the increase in ADMA blood levels. Decreased ADMA breakdown could be an alternative explanation for the increase in plasma ADMA concentrations in patients with heart failure. The predominant metabolic pathway is degradation by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA (but not SDMA) to dimethylamine and -citrulline [3,4]. To date DDAH activity is difficult to assess, however, and no data are available on (blood) DDAH activity in patients with heart failure. Dimethylarginine dimethylaminohydrolase activity could be inhibited by oxidative stress, a condition that is present in patients with heart failure [30]. Another explanation for increased plasma ADMA levels might be increased generation of ADMA from the metabolism of Hcy, because the metabolic pathways generating Hcy and ADMA are closely linked [31]. Plasma Hcy levels were in the normal range in both groups studied, however.


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Hypercholesteraemia was shown to be associated with increased plasma ADMA concentrations in vivo as well, and an inhibitory effect of LDL-cholesterol on DDAH activity was documented in vitro [19,32]. Thus increased cholesterol concentrations could theoretically contribute to the increase in plasma ADMA levels in our patients with heart failure. Serum cholesterol concentrations were not significantly different in the patients and the healthy subjects, however. Last but not least, the plasma ADMA concentration has been shown to increase with senescence [6,13], but the reason(s) for this increase is unclear. Asymmetric dimethylarginine levels in our healthy controls were high and comparable to those of elderly healthy subjects in previous reports [6,13]. In the present study both groups of subjects were matched with respect to age, however, and yet patients with heart failure had almost twice as high blood ADMA levels, indicating other mechanisms beside senescence to be responsible for the increase in the plasma ADMA concentration. Interestingly, ADMA blood levels in our patients with heart failure were comparable to those found in patients with renal disease and/or severe peripheral vascular disease [7,33]. In conclusion, the observation of a significant relationship between high blood ADMA levels and reduced renal perfusion in patients with heart failure is consistent with a causal role of ADMA in the pathophysiology of endothelial dysfunction resulting in increased renovascular tone. The latter may render the kidney susceptible for further damage. Trials with supplementation of -arginine to overcome the potential adverse (renal) effects of ADMA in these patients are warranted.

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