**4. Discussion**

The findings of the present study are as follows: (1) Serum ADMA level is increased in patients with CKD compared with healthy subjects and is associated with decreased NO and GFR. (2) Elevation of circulating serum ADMA is associated with increased Hcy and fibrinogen in CKD patients. (3) Serum NO level as dependent variable was also negatively correlated with Hcy. Our findings suggested that the ADMA levels can reflect a possible independent role in CKD pathogenesis. Increased ADMA serum levels cause persistent renal vasoconstriction and sodium retention, and contributes to the development of high blood pressure (11). In addition, it might influence NO and GFR levels and affect atherosclerosis formation.

Several studies suggested that ADMA level can be an independent risk factor for progression of CKD (3-13). Elevated ADMA reduces bioavailability of NO and induces endothelial dysfunction and may be involved in the pathophysiology of cardiovascular disease in CKD (8). ADMA fulfils many of the characteristic features of an uremic toxin (14,15). Elevation of circulated ADMA, an endogenous inhibitor of nitric oxide synthase, is an independent risk factor for cardiovascular diseases in predialysis patients with CKD (5,14,15). High ADMA levels lead to NO depletion, impaired endothelium-dependent

Urea, creatinine, calcium, phosphate, albumin, protein, high sensitive CRP (hsCRP), insulin, glucose, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride assays were determined by standard laboratory methods according to the established methodology. The serum level of fibrinogen was measured by the Clauss method using a commercial kit. All routine

Statistical analysis of the differences between groups of subjects was performed using the Kolmogorov-Smirnov and unpaired student's t-test or by the Mann-Whitney non-

Serum levels of ADMA, Hcy, creatinine, LDL-C and hsCRP were significantly (p<0.001) higher in patients with mild CKD than in healthy controls. Also, systolic and diastolic blood pressures were increased (p<0.001). There were no significant differences in levels of serum fasting blood glucose, insulin, total cholesterol, HDL-C, triglyceride, calcium and phosphate between the mild CKD and healthy controls (P>0.05). Serum NO and creatinine clearance levels were decreased in patients with mild CKD than in healthy controls (p<0.001). Clinical and laboratory data are reported in Table 1. In multiple linear regression analysis, ADMA level was negatively correlated with NO (r = -0.861; p<0.001) as shown in Figure 2A, and positively correlated with Hcy (r = 0.547; p<0.001, Figure 2B) and fibrinogen (r = 0.704; p<0.01, Figure 2C). ADMA level was positively correlated with creatinine (r=0.510;p<0.001), LDL-C (r=0.420;p<0.01), hsCRP (r=0.525;p<0.001), systolic (r=0.375; p<0.001) and diastolic blood pressure (r=0.410;p<0.001) levels. ADMA level was negatively correlated with GFR (r=-0.720;p<0.001). Also, serum NO level was negatively correlated with homocystein (r = - 0.390; p<0.001, Figure 3). We found no association between ADMA and HDL-C or other

The findings of the present study are as follows: (1) Serum ADMA level is increased in patients with CKD compared with healthy subjects and is associated with decreased NO and GFR. (2) Elevation of circulating serum ADMA is associated with increased Hcy and fibrinogen in CKD patients. (3) Serum NO level as dependent variable was also negatively correlated with Hcy. Our findings suggested that the ADMA levels can reflect a possible independent role in CKD pathogenesis. Increased ADMA serum levels cause persistent renal vasoconstriction and sodium retention, and contributes to the development of high blood pressure (11). In addition, it might influence NO and GFR levels and affect

Several studies suggested that ADMA level can be an independent risk factor for progression of CKD (3-13). Elevated ADMA reduces bioavailability of NO and induces endothelial dysfunction and may be involved in the pathophysiology of cardiovascular disease in CKD (8). ADMA fulfils many of the characteristic features of an uremic toxin (14,15). Elevation of circulated ADMA, an endogenous inhibitor of nitric oxide synthase, is an independent risk factor for cardiovascular diseases in predialysis patients with CKD (5,14,15). High ADMA levels lead to NO depletion, impaired endothelium-dependent

laboratory measurements were carried out using certified assay methods.

**3. Results** 

**4. Discussion** 

atherosclerosis formation.

parameters in either subjects with mild CKD.

parametric test as appropriate. Pearson's correlation analyses were performed.


\*P < 0.001; Data are reported as means ± SD.

BP: Blood Pressure; HDL-C: High Density Lipoprotein Cholesterol; LDL-C: Low Density Lipoprotein Cholesterol; hsCRP: High sensitive C Reactive Protein; ADMA: Asymmetric dimethylarginine

Table 1. Clinical and laboratory data of patients with CKD and healthy subjects.

The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy) on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and... 203

(c)

Fig. 2. Correlation between asymmetric dimethylarginine (ADMA) and **(A)** nitric oxide

Fig. 3. Correlation between nitric oxide (NO) and homocysteine (Hcy).

(NO), **(B)** homocysteine (Hcy), and **(C)** fibrinogen.

(a)

(a)

(b)

Fig. 2. Correlation between asymmetric dimethylarginine (ADMA) and **(A)** nitric oxide (NO), **(B)** homocysteine (Hcy), and **(C)** fibrinogen.

Fig. 3. Correlation between nitric oxide (NO) and homocysteine (Hcy).

The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy) on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and... 205

glomerular capillaries (30).

(31).

18,22).

baseline kidney function.

derived nitric oxide is a potent endothelial vasodilator which balances constrictors to regulate blood pressure and vascular tone (9). Leone et al. (35) suggested that NO may play a role in blood pressure regulation. NO is a cardiovascular protective substance because it causes vasodilation and leucocyte aggregation (10). Nitric oxide also plays a role in regulating renal sodium excretion and renin release (30). Nitric oxide, synthesised from Larginine, contributes to the regulation of blood pressure and to host defence (29). As an endogenous vasodilator it contributes to renal arteriolar tone and modulates relaxation of the mesangium, thus contributing to regulation of glomerular microcirculation. It has antiplatelet and antithrombogenic effects and thus helps prevent thrombosis within the

Clinical and experimental evidence suggest that the elevation of ADMA may cause a low production of NO (11,14-17,29,30). Synthesis of NO can be blocked by inhibition of nitric oxide synthase (NOS) activities with guanidino-substituted analogues of L-arginine such as ADMA (28). Accumulation of endogenous ADMA, leading to impaired NO synthesis, might contribute to the hypertension and immune dysfunction associated with chronic renal failure (29). Reduced bioavailability of NO, increased systemic blood pressure, endothelial cell injury and dysfunction are thought to play an important role in progressive kidney damage (7). Endothelial dysfunction due to reduced availability of NO is an early step in the course of atherosclerotic vascular disease (7). Increased ADMA blood levels may contribute to this process. In addition, NO inhibits key processes of atherosclerosis, such as monocyte endothelial adhesion, platelet aggregation, and vascular smooth muscle cell poliferation

In our study, while serum ADMA and Hcy levels were significantly higher in the patients with CKD than in healthy subject, the NO level was significantly lower. Our findings were in agreement with previous studies (7,9,10,18). Low NO is a major feature of chronic kidney diseases. We examined the relationship of ADMA with NO and with Hcy in CKD patients. In this prospective study, high ADMA level was associated with both decreased NO and increased Hcy. Similarly, Strong relationships between increased serum Hcy, fibrinogen, ADMA and decreased NO, GFR and mortality from cardiovascular events have recently been demonstrated. Several prospective clinical studies have shown that ADMA, fibrinogen, Hcy, LDL-C and other cardiovascular risk parameters are effected in patients with CKD, atherosclerosis, hypertension, diabetes and other clinical entities (14-

The major factor for high plasma ADMA levels in renal failure seems to be a decrease DDAH activity, which in turn may be due to increased oxidative stress and/or hyperhomocysteinemia (18). Recent studies show contradictory data regarding the role of hyperhomocysteinemia on cardiovascular morbidity and mortality in CKD patients (32). Rasmussen et al. (22) suggested that elevated homocysteine level is an independent predictor of cardiovascular events in patients with ESRD. Ninomiya et al. (33) suggested that baseline Hcy level showed a significantly inverse association with rate of change in kidney function during the 5 years after being adjusted for confounding factors, including

One study indicates a linkage between hyperhomocysteinemia, oxidative stress and ADMA metabolism (32). Recently, it was hypothesized that some of the deleterious effects of

vasodilation and plaque rupture with thrombus formation (8). In addition, increased ADMA level in circulation is a combined result of decreased elimination and reduced activity of ADMA catabolism by dimethylarginine dimethylaminohydrolase (DDAH) (8,9). Elevated plasma levels of ADMA in patients with end stage renal disease (ESRD) were first reported by Vallance et al. (15). Several recent studies have already indicated that elevated plasma ADMA levels could cause cardiovascular morbidity and mortality in progressive chronic kidney disease (4-13). Mihout et al. (9) demonstrated that high plasma ADMA levels contribute to the development of hypertension, oxidative stress, and interstitial and glomerular fibrosis, and peritubular capillary rarefaction. This may be involved in the decline of renal function. Serum levels of ADMA in CKD are predictive of renal survival and of cardiovascular damage. High ADMA levels are associated with endothelial dysfunction and oxidative stres (12). In the study by Young et al. (8) , there was a strong association of ADMA with prevalent cardiovascular disease and a modest association with all-cause and cardiovascular disease mortality. ADMA is strongly associated with intimamedia thickness of the carotid artery and left ventricular mass, particularly concentric left ventricular hypertrophy (11).

Coen et al. (12) suggested that ADMA levels could be influenced by the severity of hyperparathyroidsm and contribute to cardivascular death linked to parathyroid hormone (PTH) of hemodialysis patients. Another study conducted by Shi et al (5) has shown that the circulating level of ADMA is an important risk factor of LVH and predicts CVD in predialysis CKD patients.

Selcoki et al. (10) reported that ADMA level was to be one of the strongest risk markers for atherosclerosis in patients with mild and moderate CKD. Ninety percent of ADMA has been metabolized by DDAH, while the other small portion, 10 %, is excreted by urinary system. Potential mechanisms of elevated plasma ADMA levels in renal failure are increased protein methylation, increased proteolysis, impaired renal excretion and impaired metabolism by DDAH (18). These results are consistent with data from our study. Our results suggest that high ADMA level can be a significant risk factor for progression of renal dysfunction in the earlier stages of CKD.

Several recent studies found markedly elevated plasma ADMA levels not only in patients with ESRD, but also in patients with progressive CKD (2). It is of note that our results are in line with a recent study by Nakamura et al. (28), who found that elevation of serum ADMA levels play a role in the progression of atherosclerosis and CKD in high-risk patients.

Studies in both the general population and the dialysis population showed a strong and independent link between ADMA, all-cause mortality, and cardiovascular events (11,12,21,24). As a consequence, elevated serum levels of ADMA may be of relevance not only in vascular pathology but also in the pathophysiology of hypertension, and in paralel, in the development of renal damage (13).

When ADMA accumulates in CKD due to defective inactivation and excretion, it is a factor of impaired NO synthesis. The decrease in the generation of NO lead to endothelial malfunction and damage (12). Nitric oxide is an important molecule which has many physiological functions, such as mediating vasodilation, inhibiting atherosclerosis, and modulating the growth of the myocardium (5). Nitric oxide is produced from its precursor L-arginine via a reaction catalyzed by endothelial NO synthase (NOS) (8,9). Endothelium-

vasodilation and plaque rupture with thrombus formation (8). In addition, increased ADMA level in circulation is a combined result of decreased elimination and reduced activity of ADMA catabolism by dimethylarginine dimethylaminohydrolase (DDAH) (8,9). Elevated plasma levels of ADMA in patients with end stage renal disease (ESRD) were first reported by Vallance et al. (15). Several recent studies have already indicated that elevated plasma ADMA levels could cause cardiovascular morbidity and mortality in progressive chronic kidney disease (4-13). Mihout et al. (9) demonstrated that high plasma ADMA levels contribute to the development of hypertension, oxidative stress, and interstitial and glomerular fibrosis, and peritubular capillary rarefaction. This may be involved in the decline of renal function. Serum levels of ADMA in CKD are predictive of renal survival and of cardiovascular damage. High ADMA levels are associated with endothelial dysfunction and oxidative stres (12). In the study by Young et al. (8) , there was a strong association of ADMA with prevalent cardiovascular disease and a modest association with all-cause and cardiovascular disease mortality. ADMA is strongly associated with intimamedia thickness of the carotid artery and left ventricular mass, particularly concentric left

Coen et al. (12) suggested that ADMA levels could be influenced by the severity of hyperparathyroidsm and contribute to cardivascular death linked to parathyroid hormone (PTH) of hemodialysis patients. Another study conducted by Shi et al (5) has shown that the circulating level of ADMA is an important risk factor of LVH and predicts CVD in pre-

Selcoki et al. (10) reported that ADMA level was to be one of the strongest risk markers for atherosclerosis in patients with mild and moderate CKD. Ninety percent of ADMA has been metabolized by DDAH, while the other small portion, 10 %, is excreted by urinary system. Potential mechanisms of elevated plasma ADMA levels in renal failure are increased protein methylation, increased proteolysis, impaired renal excretion and impaired metabolism by DDAH (18). These results are consistent with data from our study. Our results suggest that high ADMA level can be a significant risk factor for progression of renal dysfunction in the

Several recent studies found markedly elevated plasma ADMA levels not only in patients with ESRD, but also in patients with progressive CKD (2). It is of note that our results are in line with a recent study by Nakamura et al. (28), who found that elevation of serum ADMA

Studies in both the general population and the dialysis population showed a strong and independent link between ADMA, all-cause mortality, and cardiovascular events (11,12,21,24). As a consequence, elevated serum levels of ADMA may be of relevance not only in vascular pathology but also in the pathophysiology of hypertension, and in paralel,

When ADMA accumulates in CKD due to defective inactivation and excretion, it is a factor of impaired NO synthesis. The decrease in the generation of NO lead to endothelial malfunction and damage (12). Nitric oxide is an important molecule which has many physiological functions, such as mediating vasodilation, inhibiting atherosclerosis, and modulating the growth of the myocardium (5). Nitric oxide is produced from its precursor L-arginine via a reaction catalyzed by endothelial NO synthase (NOS) (8,9). Endothelium-

levels play a role in the progression of atherosclerosis and CKD in high-risk patients.

ventricular hypertrophy (11).

dialysis CKD patients.

earlier stages of CKD.

in the development of renal damage (13).

derived nitric oxide is a potent endothelial vasodilator which balances constrictors to regulate blood pressure and vascular tone (9). Leone et al. (35) suggested that NO may play a role in blood pressure regulation. NO is a cardiovascular protective substance because it causes vasodilation and leucocyte aggregation (10). Nitric oxide also plays a role in regulating renal sodium excretion and renin release (30). Nitric oxide, synthesised from Larginine, contributes to the regulation of blood pressure and to host defence (29). As an endogenous vasodilator it contributes to renal arteriolar tone and modulates relaxation of the mesangium, thus contributing to regulation of glomerular microcirculation. It has antiplatelet and antithrombogenic effects and thus helps prevent thrombosis within the glomerular capillaries (30).

Clinical and experimental evidence suggest that the elevation of ADMA may cause a low production of NO (11,14-17,29,30). Synthesis of NO can be blocked by inhibition of nitric oxide synthase (NOS) activities with guanidino-substituted analogues of L-arginine such as ADMA (28). Accumulation of endogenous ADMA, leading to impaired NO synthesis, might contribute to the hypertension and immune dysfunction associated with chronic renal failure (29). Reduced bioavailability of NO, increased systemic blood pressure, endothelial cell injury and dysfunction are thought to play an important role in progressive kidney damage (7). Endothelial dysfunction due to reduced availability of NO is an early step in the course of atherosclerotic vascular disease (7). Increased ADMA blood levels may contribute to this process. In addition, NO inhibits key processes of atherosclerosis, such as monocyte endothelial adhesion, platelet aggregation, and vascular smooth muscle cell poliferation (31).

In our study, while serum ADMA and Hcy levels were significantly higher in the patients with CKD than in healthy subject, the NO level was significantly lower. Our findings were in agreement with previous studies (7,9,10,18). Low NO is a major feature of chronic kidney diseases. We examined the relationship of ADMA with NO and with Hcy in CKD patients. In this prospective study, high ADMA level was associated with both decreased NO and increased Hcy. Similarly, Strong relationships between increased serum Hcy, fibrinogen, ADMA and decreased NO, GFR and mortality from cardiovascular events have recently been demonstrated. Several prospective clinical studies have shown that ADMA, fibrinogen, Hcy, LDL-C and other cardiovascular risk parameters are effected in patients with CKD, atherosclerosis, hypertension, diabetes and other clinical entities (14- 18,22).

The major factor for high plasma ADMA levels in renal failure seems to be a decrease DDAH activity, which in turn may be due to increased oxidative stress and/or hyperhomocysteinemia (18). Recent studies show contradictory data regarding the role of hyperhomocysteinemia on cardiovascular morbidity and mortality in CKD patients (32). Rasmussen et al. (22) suggested that elevated homocysteine level is an independent predictor of cardiovascular events in patients with ESRD. Ninomiya et al. (33) suggested that baseline Hcy level showed a significantly inverse association with rate of change in kidney function during the 5 years after being adjusted for confounding factors, including baseline kidney function.

One study indicates a linkage between hyperhomocysteinemia, oxidative stress and ADMA metabolism (32). Recently, it was hypothesized that some of the deleterious effects of

The Effects of Asymmetric Dimethylarginine (ADMA), Nitric Oxide (NO) and Homocysteine (Hcy) on Progression of Mild Chronic Kidney Disease (CKD): Relationship Between Clinical and... 207

(eds).Little, Brown and Company, Boston 1994: 604-622.

renal disease. Kidney Int 2003;63(suppl85):S105-S110.

IgA Nephropathy. Am J Nephrol 2011; 33: 1-6.

recipients. Kidney Int 2010;77(1): 44-50.

synthesis. J Pathol 2011; 223(1) : 37–45.

binding. Clin Nephrol 2004;62(4):295-300.

Drug Discovery 2002;1(12):939-950

disease. Kidney Int 2000;58:1261-1266.

Nephrol 2009;22(5):616-622.

1992;339(8793):572-575.

2008;294(1):F1-F9.

[1] Zawada ET. Indications for dialysis. Handbook of Dialysis. Daugirdas JT, Ing TS

[2] Zoccali C, Mallamaci F, Tripepi G. Traditional and emerging risk factors in end-stage

[3] Busch M, Franke S, Miller A, et al. Potential risk factors in chronic kidney disease: EGEs,

[4] Fujimi-Hayashida A, Ueda S, Yamagishi S, et al. Association of asymmetric

[5] Shi B, Ni Z, Zhou W, et al. Circulating levels of asymmetric dimethylarginine are an

[6] Abedini S, Meinitzer A, Holme I, et al. Asymmetrical dimethylarginine is associated with

[7] Fliser D, Kielsten JT, Haller H, and Bode-Böger SM. Asymmetric dimethylarginine: A cardiovascular risk factor in renal disease? Kidney Int (Supp) 2003;(84):37–40. [8] Young JM, Terin N, Wang X, et al. Asymmetric dimethylarginine and mortality in stages 3 to 4 chronic kidney disease. Clin J Am Soc Nephrol 2009;4(6):1115–1120. [9] Mihout F, Shweke N, Big´e N, et al. Asymmetric dimethylarginine (ADMA) induces

[10] Selcoki Y, Aydn M, İkizek M, Armutcu F, Eryonucu B, Kanbay M. Association between

[12] Coen G, Mantella D, Sardella D, et al. Asymmetric dimethylarginine, vascular

[13] Kielstein JT, Böger RH, Bode-Böger SM, et al. Low dialysance of asymmetric

[14] Vallance P, Leiper J. Blocking NO synthesis: How, where and why? Nature Reviews

[15] Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous

[16] Schmidt RJ, Baylis C. Total nitric oxide production is low in patients with chronic renal

[17] Baylis C. Nitric oxide deficiency in chronic kidney disease. Am J Physiol Renal physiol

synthase inhibition. Kidney Blood Pres Res 2004;27(3):143-147.

total homocysteine and metabolites, and the C-reactive protein. Kidney Int

dimethylarginine with severity of kidney injury and decline in kidney function in

independent risk factor for left ventricular hypertrophy and predict cardiovascular events in pre-dialysis patients with chronic kidney disease. Eur J Intern Med

renal and cardiovascular outcomes and all-cause mortality in renal transplant

chronic kidney disease through a mechanism involving collagen and TGF-β1

asymmetric dimethylarginine and the severity of coronary artery disease in patients with chronic kidney disease. Turk Neph Dial Transpl 2011;20(1):58-64. [11] Kielstein JT, Simmel S, Bode-Böger SM, et al. Subpressor dose asymmetric

dimethylarginine modulates renal function in humans through nitric oxide

calcifications and parathyroid hormone serum levels in hemodialysis patients. J

dimethylarginine (ADMA)- in vivo and in vitro evidence of significant protein

inhibitor of nitric oxide synthesis in chronic renal failure. Lancet

**5. References** 

2004;66:338-347.

2010;21(5):444-8.

hyperhomocysteinemia may involve ADMA-related cardiovascular effect in CKD (18-20). Hyperhomocysteinemia, elevated plasma ADMA concentrations have first been described in patients with renal failure (18). Plasma levels of homocysteine and ADMA are elevated in patients with renal failure and both have been associated with cardiovascular events, possibly due to their negative effects on endothelial function. ADMA in methylation of homocystein plays an important role. Elevated homocysteine level is strongly related to renal function and probably due to decreased metabolic clearance (18-20). Homocysteine and ADMA are aminoacids which are biochemically linked by a common synthetic pathway. Homocysteine inhibits DDAH, the enzyme responsible for the breakdown of ADMA. Homocysteine may enhance protein degradation by destabilizing protein structure or by increasing oxidative stress, resulting in ADMA release (18).

Contraversely, Simic-Ogrizovic et al. (24) suggested that although total serum Hcy level was not found to be a predictor of overall and cardiovascular mortality, the role of hyperhomocysteinemia as risk factor for cardiovascular disease cannot be excluded in hemodialysis patients.

We found a strong association between ADMA levels and hyperfibrinogenemia, and hyperhomocysteinemia in our study. In addition, as inflammation index, CRP and fibrinogen were increased. Our results show that increased ADMA, Hcy, hsCRP and fibrinogen levels contribute to the progression of renal disease. Serum levels of ADMA and Hcy may interact and modulate the effect of each other, thus contributing to a common mechanism leading to cardiovascular diseases in CKD. These findings are similar to observations from previous studies (18-21).

The level of serum fibrinogen (an inflammation marker) is increased in CKD. Increased serum fibrinogen level independently predicts cardiac events (20). Shishehbor et al. (19) suggested that Hcy and fibrinogen levels can explain nearly 40% of the attributable mortality risk from CKD. Bostom et al. (21) suggested that Hcy, lipoprotein(a) (Lp(a)), and fibrinogen interact to promote atherothrombosis, combined hyperhomocysteinemia, hyperfibrinogenemia, and, Lp(a) excess may contribute to the high incidence of vascular disease sequelae experienced by dialysis patients, which is inadequately explained by traditional cardiovascular disease risk factors. In our present study, the serum level of LDL-C was significantly higher in the patients with CKD than in the healthy subjects. In addition, the ADMA level was positively correlated with LDL-C. The association of increased LDL-C with increased risk of coronary heart disease may be thought as a covariable in the oxidative activation of ADMA synthesis.

Descamps-Latscha et al. (23) thought that CRP, fibrinogen and advanced oxidation protein products (AOPP) levels independently predict atherosclerotic cardiovascular events in patients with CKD in the predialysis phase and might directly contribute to the uremiaassociated accelerated atherogenesis. These findings lend support to the hypothesis that accumulation of ADMA is an important risk factor for cardiovascular events in CKD (2).

Our findings suggest that high ADMA, fibrinogen and Hcy levels and NO deficiency may contribute to the process of atherosclerotic cardiovascular disease and other consequeces of uremia in predialysis patients with CKD. In addition, the ADMA level was associated with hyperhomocysteineamia and hyperfibrinogenemia.

#### **5. References**

206 Chronic Kidney Disease

hyperhomocysteinemia may involve ADMA-related cardiovascular effect in CKD (18-20). Hyperhomocysteinemia, elevated plasma ADMA concentrations have first been described in patients with renal failure (18). Plasma levels of homocysteine and ADMA are elevated in patients with renal failure and both have been associated with cardiovascular events, possibly due to their negative effects on endothelial function. ADMA in methylation of homocystein plays an important role. Elevated homocysteine level is strongly related to renal function and probably due to decreased metabolic clearance (18-20). Homocysteine and ADMA are aminoacids which are biochemically linked by a common synthetic pathway. Homocysteine inhibits DDAH, the enzyme responsible for the breakdown of ADMA. Homocysteine may enhance protein degradation by destabilizing protein structure

Contraversely, Simic-Ogrizovic et al. (24) suggested that although total serum Hcy level was not found to be a predictor of overall and cardiovascular mortality, the role of hyperhomocysteinemia as risk factor for cardiovascular disease cannot be excluded in

We found a strong association between ADMA levels and hyperfibrinogenemia, and hyperhomocysteinemia in our study. In addition, as inflammation index, CRP and fibrinogen were increased. Our results show that increased ADMA, Hcy, hsCRP and fibrinogen levels contribute to the progression of renal disease. Serum levels of ADMA and Hcy may interact and modulate the effect of each other, thus contributing to a common mechanism leading to cardiovascular diseases in CKD. These findings are similar to

The level of serum fibrinogen (an inflammation marker) is increased in CKD. Increased serum fibrinogen level independently predicts cardiac events (20). Shishehbor et al. (19) suggested that Hcy and fibrinogen levels can explain nearly 40% of the attributable mortality risk from CKD. Bostom et al. (21) suggested that Hcy, lipoprotein(a) (Lp(a)), and fibrinogen interact to promote atherothrombosis, combined hyperhomocysteinemia, hyperfibrinogenemia, and, Lp(a) excess may contribute to the high incidence of vascular disease sequelae experienced by dialysis patients, which is inadequately explained by traditional cardiovascular disease risk factors. In our present study, the serum level of LDL-C was significantly higher in the patients with CKD than in the healthy subjects. In addition, the ADMA level was positively correlated with LDL-C. The association of increased LDL-C with increased risk of coronary heart disease may be thought as a covariable in the oxidative

Descamps-Latscha et al. (23) thought that CRP, fibrinogen and advanced oxidation protein products (AOPP) levels independently predict atherosclerotic cardiovascular events in patients with CKD in the predialysis phase and might directly contribute to the uremiaassociated accelerated atherogenesis. These findings lend support to the hypothesis that accumulation of ADMA is an important risk factor for cardiovascular events in CKD (2).

Our findings suggest that high ADMA, fibrinogen and Hcy levels and NO deficiency may contribute to the process of atherosclerotic cardiovascular disease and other consequeces of uremia in predialysis patients with CKD. In addition, the ADMA level was associated with

or by increasing oxidative stress, resulting in ADMA release (18).

hemodialysis patients.

observations from previous studies (18-21).

activation of ADMA synthesis.

hyperhomocysteineamia and hyperfibrinogenemia.


**13**

*Portugal*

**Neutrophil Activation and Erythrocyte** 

*1Instituto de Ciências da Saúde da Universidade Católica Portuguesa 2Instituto de Biologia Molecular e Celular da Universidade do Porto*

**Stage 5 Chronic Kidney Disease Patients**

Anaemia is a frequent complication associated with stage 5 chronic kidney disease (CKD), and is mainly due to insufficient production of erythropoietin by the kidneys. Accumulation of uremic toxins, excessive toxic storage of aluminium in the bone marrow (Miyoshi, 2006), blood loss (either iatrogenic, from the puncture sites of the vascular access and blood sampling, or from other sources, such as the gastrointestinal tract), and premature erythrocyte destruction have also been frequently associated with anaemia in stage 5 CKD

The erythrocyte, presenting a limited biosynthesis capacity, suffers and accumulates physical and/or chemical changes, which become more pronounced with cell aging, and whenever an unusual physical or chemical stress develops (Locatelli, 2004a). Erythrocytes are physically stressed during the haemodialysis process, and metabolically stressed by the unfavourable plasmatic environment, due to metabolite accumulation, and by the high rate of haemoglobin autoxidation, due to the increase in haemoglobin turnover, a physiologic compensation mechanism triggered in case of anaemia (Lucchi, 2000; Stoya, 2002). The erythrocytes are, therefore, continuously challenged to sustain haemoglobin in its reduced functional form, as well as to maintain the integrity and deformability of the

Leukocytosis is essential as the primary host defence, and neutrophils, the major leukocyte population of blood in adults, play a primordial role. It is well known that neutrophils have mechanisms that are used to destroy invading microorganisms. These cells use oxygen-dependent and oxygen-independent microbicidal artillery to destroy and remove infectious agents (Witko-Sarsat, 2000). Activated neutrophils also undergo degranulation, with the release of several components, namely, proteases and cationic

In this book chapter we review the cross-talk between changes in erythrocyte membrane

protein composition and the release of neutrophil activation products.

**1. Introduction**

membrane.

patients (Medina, 1994; Pisoni, 2004).

proteins (Witko-Sarsat, 2000).

**Membrane Protein Composition in** 

Elísio Costa1,2, Luís Belo2,3 and Alice Santos-Silva2,3

*3Faculdade de Farmácia da Universidade do Porto*

