Endothelin-1 and Nitric Oxide in Patients on Chronic Hemodialysis

Abstract

Aim. To establish the role of endothelin-1 and nitric oxide in the pathogenesis of hypertension in patients on chronic aemodyalisis by correlating endothelin-1 and NO plasma concentrations to the level of arterial hypertension with respect to angiotensin-converting enzyme (ACE) inhibitor therapy. Methods. We determined plasma concentrations of endothelin-1 and NO in patients on chronic hemodialysis (CHD) before and after hemodialysis treatment. The study included 30 CHD patients and 20 healthy participants as controls. Correlation to blood pressure was determined, as well as the effect of ACE inhibitors on the relationship between both endothelin-1 and NO in correlation with arterial hypertension. Main findings.Endothelin-1 plasma concentration was significantly higher in CHD patients before hemodialysis treatment than in healthy controls. Endothelin-1 plasma concentration was also significantly higher in CHD patients after hemodialysis than in healthy controls. There was a significant decrease in endothelin-1 plasma concentration after hemodialysis in comparison with its values before hemodialysis. In CHD patients, a positive correlation was found between endothelin-1 plasma concentration and systolic blood pressure after hemodialysis, irrespective of ACE inhibitors therapy. In CHD patients taking ACE inhibitors, systolic blood pressure increased with increasing endothelin-1 plasma concentration before as well as after hemodialysis. In patients taking ACE inhibitors, there was a tendency for diastolic blood pressure to increase with an increase in endothelin-1 plasma concentration after hemodialysis and to decrease with an increase in NO plasma concentration. Conclusion. NO and endothelin-1 play a significant role in etiology of the hemodynamic changes of blood pressure during the dialysis.

Keywords:

• ACE inhibitors
• endothelin-1
• hemodialysis
• hypertension
• nitric oxide

INTRODUCTION

Hypertension in patients on chronic hemodialysis (CHD) is a multifactorial condition. Traditionally, there are two types of hypertension in CHD patients.[1],[2] The first type is characterized by normal to low plasma renin activity and the possibility of decreasing high blood pressure by regulating body weight and reducing salt intake. This type of hypertension is called volume hypertension. The other type is characterized by high plasma renin activity and an inability to regulate blood pressure by reduced salt intake and normalization of body weight. This type of hypertension is called renin-dependent hypertension.[3],[4] Recently, increased consideration has been given to the influence of endothelin-1 and nitric oxide (NO) in the pathogenesis of hypertension in CHD patients. Vascular endothelium plays a central role in the regulation of vascular tone via production of vasoactive substances, including endothelin-1 as a vasoconstrictor and NO as a vasodilator. Endothelin-1 increases the activity of sympathetic nervous system and is the most powerful vasoconstricting substance known so far.[5–11] It increases arterial blood pressure, reduces renal plasma flow and glomerular filtration, and decreases natriuresis and diuresis.[12–15] In the glomeruli affected by sclerosis, endothelial injury leads to the increased secretion of endothelin-1 and consequent vasoconstriction, increased intraglomerular pressure, and decreased glomerular filtration. NO is a free radical involved in various physiological processes. NO leads to vasodilatation and inhibits the proliferation of vascular smooth muscle as well as the adhesion and aggregation of blood platelets. Produced by NO synthase from substrate L-arginine in vascular endothelial cells, NO acts as a potent vasodilator contributing to the regulation of vascular tone.[16–18] In the uremic condition, endothelial dysfunction and reduced production and effect of NO have been observed.

Decreased NO levels are the result of the lack of L-arginine due to the loss of functional renal mass.[19–21] Increased levels of endogenous NO synthase inhibitors, such as asymmetric dimethylarginine (ADMA), are also found.[19],[22–24] Circulating levels of NO synthase inhibitors accumulate in terminal stages of renal failure, when changes in the blood pressure in patients on hemodialysis are also observed.[25],[26] In patients with renal failure, the concentration of L-arginine analogues increases. L-arginine analogues competitively inhibit NO synthase, decrease L-arginine plasma concentration, and may induce arterial hypertension. They also lead to immune system damage due to impairment of NO production in the endothelium and macrophages. When renal mass in glomeruli is reduced, inflammatory mediators are increasingly secreted as well as platelet derived growth factor (PDGF) and transforming growth factor β (TGF-β). PDGF and TGF-β are potent inhibitors of NO synthase, and they block interleukin-1B-induced mRNA in a dose-dependent manner for inducible NO synthase in mesangial cells.[25],[26] The most recent results of in vitro and in vivo studies on rats have shown that reduced renal mass leads to an increased synthesis of a potent vasoconstrictor, endothelin-1, which then reduces the production of NO.[27],[28]

The aim of our study was to determine and compare plasma concentration of endothelin-1 and NO in CHD patients before and after hemodialysis treatment with those in healthy controls, and to correlate endothelin-1 and NO plasma concentrations to the level of arterial hypertension with respect to angiotensin-converting enzyme (ACE) inhibitor therapy.

MATERIALS AND METHODS

The study included 30 CHD patients, 18 men and 12 women (age range: 50–80 years; mean: 65) with various primary diseases (see Table 1). Half of the CHD patients were taking ACE inhibitors for the treatment of hypertension, whereas the other half did not take these medications. The control group included 20 healthy participants (12 men and 8 women) aged 63 years on average. Inclusion criteria were the absence of known kidney disease, hypertension, heart disease, diabetes mellitus, and systemic or malignant disease. There was no significant difference in the age and sex composition between the patient and control group. All study participants underwent a clinical examination and kidney ultrasound examination (renal size). Hemoglobin and creatinine serum concentration were also determined. Endothelin-1 and NO were measured before and after hemodialysis. In all patients, the dose of erythropoetin (see Table 2) and the type of dialysis membrane were determined. Half of them used Fresenius F6, and the other half used Fresenius F7 membrane. The data on ACE therapy and concomitant antihypertensive therapy were collected from patient medical records (see Table 3). The nature of the study was explained, and all subjects gave written consent to participate. The approval from Ethics Committe of Mostar University School of Medicine was obtained.

Table 1 Chronic hemodialysis patients regarding primary disease

Primary disease	Diabetic nephropathy	Hypertensive nephropathy	Polycystickidney disease	Chronic pyelonephritis	Unknown
Number	13	8	2	1	6
%	43.3%	26.7%	6.7%	3.3%	20%

Table 2 Chronic hemodialysis patients regarding erythropoetin dose

Eritropoetin doses /week	0	2500	5000	7500	Total
Number	4	9	11	6	30
%	13.3%	30.0%	36.7%	20.0%	100%

Table 3 Chronic hemodialysis patients regarding ACE inhibitors and concomitant antihypertensive therapy

	Patients taking ACE inhibitors	Patients not taking ACE inhibitors
Number	15	15
Concomitant therapy with Ca-blockers	5	5
Concomitant therapy with beta-blockers	0	1

Endothelin-1 was determined by enzyme-linked immunosorbent assay (ELISA). Endothelin test kit used in this study was an enzyme immunoassay designed for direct determination of endothelin in biological fluids (Endothelin ELISA-Enzyme-Linked Immunoasorbent Assay, The Next Generation, Cat. No. BI-20052, 12 × 8 test, Biomedica Gruppe, Wien, Austria).[7] NO was determined from NO₂, a stable product of NO, using Griess reaction and colorimetric determination of the concentration of colored product. Nitrites in urine were also determined with Griess reagent. Nitrite concentration was determined from the absorbance at 540 nm in the reagent solution.[29]

Statistical Analysis

Normal distribution of data was determined with Kolmogorov-Smirnov goodness-of-fit test. The significance of differences in interval variables was tested with Mann-Whitney non-parametric test if two groups of data were independent, whereas Wilcoxon non-parametric test was applied to compare dependent samples. Correlation between two interval variables or an interval and binary variable was determined by Spearman non-parametric correlation coefficient (r_s); significance of correlation was also calculated.

A p values < 0.05 was considered significant. Values were expressed as means +/− SD.

RESULTS

Plasma concentration of endothelin-1 was significantly higher in patients before hemodialysis than in healthy controls (17.02 pg/mL vs. 8.10 pg/mL, respectively; p < 0.001). In addition, plasma concentration of endothelin-1 was significantly higher in patients after hemodialysis (13.53 pg/mL) than in healthy controls (p < 0.001). There was a significant decrease in endothelin-1 plasma concentration in CHD patients after hemodialysis treatment in comparison with its values before hemodialysis (p = 0.001). NO plasma concentration in CHD patients before hemodialysis treatment was 5.66 μmol/L (i.e., not significantly different from NO plasma concentration of 5.88 μmol/L in healthy controls; p = 0.630). The average value of blood pressure in CHD patients and healthy controls was 142/82 mm Hg and 122/81 mm Hg, respectively (see Table 4).

Table 4 Plasma endothelin-1 (ET-1) and nitric oxide (NO) concentrations in patients on chronic hemodialysis (CHD) before and after dialysis and healthy controls

Parameter	Group of subjects		p
	CHD patients (n = 30)	Controls (n = 20)
Plasma endothelin-1 (pg/mL)
Before dialysis	17.0230	8.1030	0.001
After dialysis	13.5333		0.001
p	0.001
Plasma NO (μmol/L)
Before dialysis	5.6640	5.8875	0.63
After dialysis	4.2643		0.001
p	0.11

Mann-Whitney U test; Wilcoxon test.

*reaching statistical significance using tests for dependent and independent samples.

In all patients (i.e., those taking or not taking ACE inhibitors), a positive correlation was found between endothelin-1 plasma concentration and systolic blood pressure after hemodialysis (see Figure 1). There was an obvious tendency for systolic blood pressure to increase with an increase in endothelin-1 plasma concentration after hemodialysis treatment, as shown by the squared regression coefficient of R² = 0.1062 in linear regression analysis. Spearman's non-parametric correlation showed a statistically significant, positive correlation (r_s = 0.38; p = 0.030).

Figure 1. Correlation between systolic blood pressure and plasma concentration of endothelin-1 after hemodialysis in all patients irrespective of ACE inhibitor therapy.

Before hemodialysis, systolic blood pressure in patients taking ACE inhibitors increased with an increase in endothelin-1 plasma concentration. A linear model had a squared regression coefficient value of R² = 0.1063. Spearman's non-parametric correlation coefficient showed a statistically significant positive correlation (r_s = 0.28, p = 0.310). After hemodialysis, there was a tendency for systolic blood pressure in patients taking ACE inhibitors to increase with an increase in endothelin-1 plasma concentration (see Figure 2). In these patients, correlation regression analysis showed a pronounced tendency for systolic blood pressure to increase when endothelin-1 plasma concentration reached high values. There was a positive regression trend, following a linear model indicated by a high value of squared regression coefficient, R² = 0.06643, in these patients. Spearman's correlation coefficient was r_s = 0.842 (p < 0.001; see Figure 2). In patients taking ACE inhibitors, there was a tendency for diastolic blood pressure to increase with increasing endothelin-1 plasma concentration after hemodialysis, as shown by a regression coefficient of R² = 0.3343 for squared model. Correlation and regression analysis showed a tendency for systolic blood pressure values to decrease with an increase in NO plasma concentration before hemodialysis in patients taking ACE inhibitors, with squared regression coefficient of R² = 0.0961 for squared model indicating a negative correlation. Spearman's correlation coefficient was r_s= −0,271 (P = 0.140). Correlation and regression analysis showed a slight tendency for diastolic blood pressure values to decrease with an increase in NO plasma concentration (r_s = 0.15, p = 0.05), which was also indicated by regression coefficient of R² = 0.1186 for squared model (see Figure 3).

Figure 2. Correlation between systolic blood pressure and plasma concentration of endothelin-1 after hemodialysis in patients taking ACE inhibitors.

Figure 3. Correlation between diastolic blood pressure and plasma concentration of NO before hemodialysis in all patients irrespective of ACE inhibitor therapy.

In patients taking ACE inhibitors, correlation and regression analysis showed a statistically significant decrease in systolic blood pressure before hemodialysis, which correlated with the increase in NO plasma concentration (Spearman's r_s = 0.579, p = 0.020). The value of squared regression coefficient for a squared model was R² = 0.4336 (see Figure 4).

Figure 4. Correlation between systolic blood pressure and plasma concentration of NO before hemodialysis in patients taking ACE inhibitors.

In patients taking ACE, correlation and regression analysis indicated a slight tendency for diastolic blood pressure to decrease before hemodialysis as NO plasma concentration increased (r_s = -0.36; p = 0.050). This was also shown by the regression coefficient value of R² = 0.0828 for a linear model (see Figure 5).

Figure 5. Correlation between diastolic blood pressure and plasma concentration of NO before hemodialysis in patients taking ACE inhibitors.

No significant correlation was found betwen blood pressure, endothelin-1 levels, or NO levels regarding the erythropoetin therapy.

DISCUSSION

We investigated the correlation between arterial hypertension and endothelin-1 plasma concentration in CHD patients before and after hemodialysis. We found that endothelin-1 plasma concentration was higher in CHD patients than in healthy controls. A decrease in endothelin-1 plasma concentration after hemodialysis was also observed. These results are in accordance with the findings of other authors, who observed that endothelin-1 plasma concentration and big ET-1 were increasing with the progression of renal failure.[5],[30]

In our study, the patients were divided in two groups, one consisting of patients taking ACE inhibitors and the other consisting of patients not taking ACE inhibitors. In patients taking ACE inhibitors, we noticed a positive correlation between endothelin-1 plasma concentration and systolic blood pressure values before and after hemodialysis. However, in patients not taking ACE inhibitors, there was a negative correlation between endothelin-1 plasma concentration and systolic blood pressure. In patients taking ACE inhibitors, we observed a positive correlation between endothelin-1 plasma concentration and diastolic blood pressure. Although we did not include renin-angiotensin system and the effects of ultrafiltration in this study, our results support the idea that ET-1 could influence on blood pressure in hemodialysis patients.

In glomeruli affected by sclerosis, damage to the endothelium leads to an increased secretion of endothelin-1, consequent vasoconstriction, increase in intraglomerular pressure, and decrease in glomerular filtration. The most recent in vitro and in vivo data on rats have shown that a decreased renal mass leads to an increased synthesis of a potent vasoconstrictor, endothelin-1, which decreases the production of NO.[27],[28]

The endothelins are powerful vasoconstrictor peptides of which endothelin-1 is the major isoform. Endothelin-A (ETA) and endothelin-B (ETB) receptors are found in vascular smooth muscle, where their activation modulates vasoconstriction. In one study, Goddard et al. have shown that endothelin receptor antagonists used in patients with chronic renal failure decreased blood pressure.[31] They conducted a randomized, placebo-controlled, double-blind study comparing ET receptor antagonists of ETA and ETB, given alone and in combination, in eight hypertensive chronic renal failure patients and eight matched healthy controls. ETA antagonist, alone and in combination with ETB antagonist, reduced blood pressure in chronic renal failure versus placebo.

Ottosson-Seeberger et al. investigated a relationship between blood pressure and endothelin-1 plasma concentration, but found no statistically significant correlation. They observed a significant decrease in metabolic degradation of endothelin-1 in the uremic condition and found that the half-life of exogenous endothelin-1 was increased in hemodialyzed patients. However, they found no correlation between endothelin-1 concentration in subendothelial space and its plasma concentration in peripheral veins.[32] Hand et al. conducted a functional investigation of correlation between endothelin-1 and blood pressure in CHD patients and found a decreased influence of endothelin-1 on the vascular tone.[33]

Demudh et al., who measured endothelin-1 plasma concentration and monitored cardiovascular changes in CHD patients, reported a significant correlation between endothelin-1 plasma concentration and cardiovascular events.[34] Similar results were obtained by Nabokov et al., who investigated atherosclerosis and renal failure on a rat model and found a correlation between endothelin-1 plasma concentration and atherosclerotic changes.[35]

Vascular endothelium plays an important role in the tone of blood vessels and atherosclerosis prevention. In the uremic condition, endothelial dysfunction and decreased production and activity of NO was found.[36],[37] A decreased NO concentration was the result of the lack of L-arginine due to the loss of functional renal mass.[19–21] An increased concentration of endogenous NO synthase inhibitors, such as ADMA, was also observed.[19],[20],[22–24] Circulating levels of NO synthase inhibitors accumulate in the terminal stages of renal failure, and the blood pressure in patients on hemodialysis changes.[25],[26]

Haynes et al. have shown that the systemic administration of nitric oxide synthase inhibitor N^G-mono-methyl-L-arginine (L-NMMA) to humans causes vasoconstriction and increases arterial pressure, confirming that nitric oxide is generated physiologically to oppose vasoconstriction influences in resistance vessels. In this study, eight healthy men received an inhibitor of nitric oxide synthase (L-NMMA) and saline placebo intravenously. L-NMMA significantly increased mean arterial pressure and total peripheral resistance. L-NMMA also significantly decreased plasma nitrate and urinary excretion of nitrate.[38]

In patients with renal failure, the increased concentration L-arginine analogues, which competitively inhibit NO synthase, decreases the plasma level of L-arginine and may induce arterial hypertension. When renal mass in the glomeruli is decreased, inflammatory mediators, PDGF, and TGF-β are secreted in large amounts. PDGF and TGF-β are potent inhibitors of NO synthase and block in a dose-dependent manner IL-1B-induced mRNA for inducible NO synthase in mesangial cells.[25],[26]

NO deficiency has been reported in children and adults with chronic renal disease.[39] In their study, Schmidt et al. have shown that total NO production was low in patients with chronic renal failure, and they concluded that this NO deficiency may contribute to the increased blood pressure that occurs in end stage renal disease. These authors have discussed the causes of deficiency in total NO production in chronic renal failure and concluded that the lack of functional renal mass will compromise the main endogenous sources of arginine generation and could lead to arginine deficiency and impaired NO synthesis.[40] There is also evidence that circulating levels of endogenous NO synthase inhibitors increase in renal failure. Of these NO synthase inhibitors, ADMA acts as a potent nonselective NO synthase inhibitor, whereas symmetric dimethylarginine (SDMA) is ineffective.[41]

In several animal models of renal disease, the administration of L-arginine decreased the degree of glomerulosclerosis, infiltration of macrophages, and tubulointerstitial changes, probably due to increased NO synthesis. L-arginine/NO pathways play an important role in hypertension, renal disease, and atherosclerosis. These pathways interact with renal renin-angiotensin system, endothelins, cytokines, and nuclear factor kappa B(NF-κB).[42]

In rats with subototal nephrectomy, 24-hour NO excretion is reduced,[43–45] suggesting reduced total NO production. Reduced urinary NO excretion has also been reported in children with chronic renal disease in adults with IgA nephropathy and hypertension and in adults with chronic renal disease by a variety of causes.[23],[39],[46]

In our study, CHD patients did not have lower NO plasma concentration than did healthy controls. We noticed a negative correlation between NO plasma concentration and systolic blood pressure. In CHD patients before hemodialysis, a negative correlation was observed between NO plasma concentration and diastolic blood pressure. In CHD patients taking ACE inhibitors, a negative correlation between NO plasma concentration and diastolic blood pressure was found both before and after hemodialysis. Although endotelin-1 concentration was lower after dialysis, the concentration of NO did not change after dialysis. One possible explanation for the difference between NO and endothelin-1 could be the use of ACE inhibitors in the treatment of hypertension, as ACE inhibitors could increase NO level.

NO metabolism is altered in patients on hemodialysis. Circulating concentrations of NO synthase inhibitors accumulate in terminal stages of renal failure and blood pressure during hemodialysis changes. These changes in blood pressure correlate with NO concentration. Higher NO concentrations appear with a decrease in blood pressure, whereas lower NO concentrations appear with an increase in blood pressure during hemodialysis.[19],[20] In patients on hemodialysis, NO plasma concentration was found to be high (due to a complete loss of renal clearance), although the total NO production was low.[47] CHD patients known to suffer from atherosclerosis have higher concentrations of ADMA in comparison with patients on hemodialysis without verified atherosclerosis.[48]

In conclusion, the etiology of dialysis-induced hypertension remains speculative. There is mounting evidence that nitric oxide (NO) and endothelin (ET-1) may play a vital role in etiology of the hemodynamic changes of blood pressure during the dialysis. Our results, as well as those of other investigators, have shown that nitric oxide and endothelin balance may be involved in the pathogenesis of dialysis hypertension. Further studies will be required in order to definitely ascertain the role of endothelin-1 and nitric oxide in the pathogenesis of hypertension in chronic renal failure.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.