**The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy) on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and Biochemical Parameters**

A. Atamer1, S. Alisir Ecder2, Y. Atamer3, Y. Kocyigit4, N. Bozkurt Yigit5 and T. Ecder6 *1Haydarpasa Training and Research Hospital, Department of Internal Medicine, Division of Gastroenterology, Istanbul 2Goztepe Training and Research Hospital, Department of Internal Medicine, Division of Nephrology, Istanbul 3Dicle University Medical Faculty, Department of Clinical Biochemistry, Diyarbakir 4Dicle University Medical Faculty, Department of Physiology, Diyarbakir 5Yalova University, Termal Vocational School, Department of Physical Medicine and Rehabilitation, Yalova 6Istanbul University, Istanbul Medical Faculty, Department of Internal Medicine, Division of Nephrology, Istanbul, Turkey* 

#### **1. Introduction**

196 Chronic Kidney Disease

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of immune and renin-angiotensin systems reduces the pace of glomerulosclerosis

antiproteinuric approach with mycophenolate mofetil fully suppresses progressive nephropathy of experimental animals. J Am Soc Nephrol 10: 1542-1549, 1999.

> Chronic kidney disease (CKD) is a syndrome characterized by the progressive and irrevocable loss of nephrons due to several diseases. Chronic kidney disease has a varying spectrum ranging from normal renal function to uremic syndrome. Actually, the stages of renal failure have interpenetrated each other and it is not possible to draw a clear line between them. The most important reason of mortality and morbidity of patients with CKD are cardiovascular diseases and atherosclerotic complications; cardiac insufficiency 15%, myocardial infarction 10%, pericarditis 3% (1, 2). Development of vascular injury in CKD is caused by both classic (Framingham) risk factors (hypertension, dyslipidemia, smoking, diabetes mellitus) and CKD specific factors (anaemia, secondary hyperparathyroidism etc). Besides, there are papers reporting that recently defined potential risk factors such as homocysteine (Hcy), C-reactive protein (CRP), interleukin-6 (IL-6), fibrinogen, soluble intracellular adhesion molecule (sICAM-1), asymmetric dimethyl arginine (ADMA), cardiac specific troponin-I (cTnI), advanced glycation endproducts have a role in the development of accelerated atherosclerosis seen in patients with CKD (2-13). Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase and it is a guanidine analogue of L-arginine aminoacid detectable in human urine

The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy) on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and... 199

early stage CKD and to evaluate the relation of these parameters with each other.

**2. Material and methods** 

**2.1 Subjects** 

excluded.

participant.

**2.2 Methods** 

autoinjecting onto the column.

The aim of this study was to investigate the role of uremia-related cardiovascular risk factors, such as ADMA, NO, Hcy and fibrinogen, in the pathogenesis and progression of

This prospective study was carried out in 65 untreated mild chronic kidney disease (35 men and 30 women; mean age 55.2 ± 9.6 years) and 65 healthy control subjects with matched age, sex and body mass index (BMI). The creatinine clearance was calculated by the Cockcroft-Gault Formula (25). Patients having creatinine clearance less than 75 ml/min were considered to have mild CKD. Body mass index was determined as weight divided by the square of height (kg/m2). The underlying causes of CKD were glomerulonephritis (n=17), interstitial nephropathy (n=12), autosomal dominant polycystic kidney disease (n=13), chronic pyelonephritis (n=7) and urological problems (n=5). No cause was identified in 11 cases. The exclusion criteria were diabetes mellitus, active hepatitis, malignancy, smoking and infectious disease. Patients using vitamin supplements were also

The study protocol was approved by the Ethics Committee of the Dicle University School of Medicine (Diyarbakir, Turkey) and written informed consent was obtained from each

In all patients, venous blood samples were drawn between 7:00 AM after a 12-h fastened, and the serum was frozen at -70 C in aliquots until biochemical analysis were performed. *ADMA Measurement:* ADMA was measured by HPLC according to the method described by Chen et al. (26). Mobile phases consisting of 50 mM sodium acetate (pH 6.8), methanol and tetrahydrofuran (THF) (A, 82:17:1; B, 22:77:1) were used. All separations were performed at 270C and at a flow-rate of 1.0 ml/min. The wavelengths of fluorescence detector were set at 338 nm and 425 nm for excitation and emission, respectively. 20 mg of 5-sulfosalicylic acid (5-SSA) was added to 1 ml plasma, and the mixture was left in an ice bath for 10 min. The precipitated protein was removed by centrifugation at 2000 g for 10 min. o-Phthaldialdehyde (OPA) (10 mg) was dissolved in 0.5 ml of methanol, and 2 ml of 0.4 M borate buffer (0.4 M boric acid adjusted to pH 10.0 with potassium hydroxide) and 30 μl of mercaptoethanol were added. The derivatization was performed by mixing 10 μl of sample or working standard solution and 100 μl of OPA reagent and reacting for 3 min before

*NO Measurement:* The serum level of NO was measured using a colorimetric method based on the Griess reaction (27), in which nitrite is reacted with sulphanilamide and N-(1 naphthyl) ethylenediamine to produce an azo dye that can be detected at 540 nm. This was

*Hcy Measurement:* Serum level of Hcy was measured using HPLC with fluorescence

carried out after enzymatic reduction of nitrate to nitrite with nitrate reductase.

detection (Shimadzu RF-10A fluorescence detector; Shimadzu Co., Kyoto, Japan).

and plasma synthesized from endothelial cells (Figure 1). It is shown that high ADMA level increases the cardiovascular incident risk by 34% and mortality risk by 52% (4-8). Increased ADMA concentration has a high prevalence in hyperhomocysteinemia, coronary artery diseases, hypercholesterolemia, diabetes mellitus, hypertension, preeclampsia, peripheral arterial occlusive disease, impaired renal function and other diseases (7,9,10). Reduced nitric oxide (NO)-dependent vasodilation is regarded as an early indicator of atherosclerotic diseases (7,14). It is documented that adult patients with renal failure have 2-6 times higher ADMA than healthy subjects due to reduced renal excretion and reduced enzymatic degradation (15). NO is synthesized from L-arginine via NO synthase enzyme. NO inhibition decreases endothelial derived vasodilation and increases vascular resistance. Reduced NO availability can occur in patients with CKD. Moreover CKD can contribute to the accelaration of hypertension and cardiovascular complications. It appears that the increase in endogenic NO inhibitors like ADMA plays a major role in this process (11, 15- 17). It has been shown that Hcy stimulates ADMA formation and plasma ADMA levels elevate in humans and animals by hyperhomocysteinemia (18-20). Increased serum Hcy level in adult CKD patients is an independent risk factor for cardiovascular system mortality. Elevated ADMA and hyperhomocysteinemia may be due to decreased renal excretion (18-22). It is reported that ADMA formation may be related with Hcy metabolism (18,19). It was found that there is a significant interaction of serum fibrinogen and CKD with respect to risk of both fatal/nonfatal coronary events and death (20–24).

The aim of this study was to investigate the role of uremia-related cardiovascular risk factors, such as ADMA, NO, Hcy and fibrinogen, in the pathogenesis and progression of early stage CKD and to evaluate the relation of these parameters with each other.
