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Abstract
End-stage renal disease and its treatment with continuous ambulatory peritoneal dialysis (CAPD) can affect almost all organs and organ systems including vascular endothelium. Consequently, disturbance in the production of vasoactive substances endothelin-1 (ET-1) and nitric oxide (NO) occurs in these patients. There are only a small number of studies that investigated the impact of long-term CAPD on imbalance in production of vasoactive substances ET-1 and NO among these patients. Therefore, our study aimed to investigate the impact of duration of CAPD on potential overproduction of ET-1 and NO in uremic patients. This study included 23 uremic patients [10 males, mean age: 56.3 (±16.2) years] treated with CAPD. All studied patients were further divided into subgroups, groups A and B. Group A included patients on treatment with CAPD <5 years, and group B included those on treatment longer than five years. Our results showed that serum levels of these vasoactive substances are significantly higher among patients treated with CAPD longer than five years (ET-1: 51.24 ± 32.11 vs. 139.53 ± 42.42; NO: 15.50 ± 2.57 vs. 26.57 ± 5.96, respectively). We concluded that imbalance in production of vasoactive substances is present in long-term CAPD treatment and this imbalance can lead to disturbance in the local blood flow control.
Introduction 
Patients with end-stage renal disease (ESRD) are at a higher risk of developing arterial disease and its adverse health outcomes compared pared to individuals in the general population with normal renal function.[1] In this population, although classic atherosclerosis risk factors (e.g., age, smoking, diabetes, hypertension, and hyperlipidemia) are common, ESRD itself represents a unique and isolated risk factor for arterial disease.[2] Vascular endothelium is not just a mechanical barrier in blood vessel but is an endocrine organ as well, which produces many substances, out of which some have vasoactive effects.[3],[4] From physiological point of view, endothelin-1 (ET-1) is a 21-amino acid peptide that has been characterized as the most potent vasoconstrictor in the human body. ET-1 is produced by almost every cell type but especially by endothelial cells in the kidney. However, evidence shows that production of ET-1 is also done by cells of the inner medullary collecting duct as well.[5],[6],[7] Nitric oxide (NO) is a vasoactive substance produced by vascular endothelium with vasodilatory effect.[8] In ESRD patients, endothelial dysfunction that causes either decreased production or overproduction of ET-1 and NO was found.[9],[10] Patients with ESRD require treatment with renal replacement therapies with hemodialysis or continuous ambulatory peritoneal dialysis (CAPD). Dialysis itself has negative effects on almost all organs and organ systems. Some studies confirmed that patients treated with CAPD have endothelial damage compared to healthy individuals and consequently produce an imbalance in the production of vasoactive substances ET-1 and NO.[11] There are only a small number of studies which investigated the impact of long-term CAPD on imbalance in the production of vasoactive substances, ET-1 and NO, in these patients. The aim of this study was to investigate the impact of duration of CAPD treatment on the potential overproduction of ET-1 and NO among ESRD patients.
Materials and Methods This was a prospective study which included ESRD patients treated with CAPD. The study included 23 patients [10 males with mean age of 56.3 (±16.2 years] who were treated with CAPD. Dialysis solution was changed three times per day and patients were trained to do it by themselves or it was carried out at the institute under the supervision of a member of the medical staff. This study was approved by the local human ethics committee, and study participants supplied written, informed consent before enrollment in the study. At the beginning of this study, every patient was thoroughly examined and chest X-ray was used to exclude the presence of any pulmonary disease. We also did not find any other comorbidity that could induce ventilatory failure in studied patients. None of these patients had hemodynamic instability during CAPD.
All studied patients were further divided into subgroups (group A and group B). Group A included patients treated with CAPD <5 years, and group B included patients treated with CAPD longer than five years.
Blood samples from all observed patients were taken from the cubital vein just before emptying of the peritoneal cavity.
Measurement of endothelin-1 serum levels
Serum was separated from the whole blood using a heated bath at 37°C. Activity of serum ET-1 was measured with the EIA methodology which is based on an immunometric assay, the so-called “sandwich technique.” Measurement was performed using a computer-based ELISA reader (ELx 800 Universal Microplate Reader, BioTek Instruments, Inc, USA) with a wavelength of 405 nm. We used a prepared enzyme kit (ET-1; EIA kit – IBL, Hamburg, Germany).
Measurement of NO serum levels
The NO level in whole blood is determined by measuring nitrite and nitrate (NO32- u NO32-) production using classical colorimetric reaction (Griess). Blood samples for the determination of NO concentration were diluted 1:1 (vol/vol) with 0.9% saline, protein precipitated using 30% ZnSO4, 0.05 mL per mL of blood, and centrifuged at 700 g for 10 min and frozen at –20°C. NO22- concentration in serum was determined by classic colorimetric Griess reaction. Briefly, equal volumes of samples and Griess reagent (sulfanilamide and naphthalene-ethylenediamine dihydrochloride) were mixed at room temperature. After 5 min, the absorbance was measured at 546 nm using spectrophotometer. The concentration of nitrite was determined by a standard curve prepared with sodium nitrite.
Statistical Analysis The results were processed using a standard statistical method (Student’s t-test for small independent samples), and the results were shown as mean ± standard deviation. We tested the significance of differences in mean values between studied groups with the aim of monitoring changes in respiratory function parameters as well as enzymatic activity. The obtained results were stored in table (MS Excel 2013), and the IBM SPSS Statistics for Windows version 21.0 (IBM Corp., Armonk, NY: IBM Corp.) was used for all statistical analysis. We considered the value of P <0.05 as statistically significant.
Results The basic demographic and clinical characteristics of 23 individuals with ESRD treated with CAPD are presented in [Table 1].
Table 1: Basic demographic and clinical characteristics of studied patients.[Figure 1] shows the comparison of serum levels of ET-1 in patients with ESRD treated by CAPD in both studied groups (groups A and B). The mean serum level (±SD) of ET-1 in group A was 51.24 ± 32.11 and in group B was 139.53 ± 42.42. Serum ET-1 levels were significantly higher (P <0.01) in patients treated by CAPD longer than five years (group B).
Figure 1: Mean serum levels of ET-1 in all studied subjects (groups A and B).[Figure 2] shows the comparison of serum levels of NO in patients with ESRD treated by CAPD in both studied groups (groups A and B). The mean serum level (±SD) of NO in group A was 15.50 ± 2.57 and in group B was 26.57 ± 5.96. Serum NO levels were significantly higher (P <0.01) in patients treated by CAPD longer than five years (group B).
Figure 2: Mean serum levels of NO in all studied subjects (groups A and B). Discussion The main finding of this study is that prolonged duration of CAPD affects the serum levels of ET-1 and NO. Serum levels of these vasoactive substances are significantly higher in patients treated with CAPD longer than five years. Due to the fact that the number of studies which tested the influence of length of CAPD to serum levels of ET-1 and NO is small, the comparison of our data to other publications is quite difficult.
Some earlier studies and authors showed that patients treated with regular CAPD had significantly higher levels of ET-1 compared to healthy subjects.[12],[13],[14],[15] At present, it is not well known whether the high plasma levels of ET-1 and big ET-1 in dialyzed patients are caused by increased production or decreased degradation or both. In addition, it is unclear whether CAPD reduces or increases plasma concentrations of ET-1 and big ET-1, because previous studies have demonstrated conflicting results.[16] Some authors showed that serum levels of ET-1 were higher in CAPD patients compared to the control group, although statistical significance was not reached.[17] These results support conflicting and contradictory findings regarding serum levels of ET-1 in CAPD patients and healthy volunteers. Despite all noted studies, most authors state that ET-1 values are higher in patients treated with CAPD compared to healthy individuals.[15]
Physiological explanation for these results can be found in the fact that in glomeruli affected by sclerosis, damage to the endothelium leads to an increased secretion of ET-1. An increase in secretion of ET-1 leads to consequent vasoconstriction, an increase in intra-glomerular pressure, and a decrease in glomerular filtration.[18],[19]
In addition, a slight increase of ET-1 in the peritoneal cavity, lower elimination of ET-1 via peritoneal membrane, and application of human recombinant erythropoietin may be responsible for increase in serum levels of ET-1 in these patients.[20],[21],[22],[23]
The contradiction of the results concerning serum levels NO in CAPD patients has been supported by a large number of authors and studies. One group of authors stated that serum levels of NO in ESRD patients treated with CAPD were significantly lower in comparison to healthy subjects, while others reported that there was no statistically significant difference in NO serum levels between these observed groups or that NO serum levels were significantly higher in ESRD patients treated with CAPD.[17],[24],[25],[26]
Reduction of renal parenchyma can contribute to decreased concentration of L-arginine, and this physiological mechanism can explain lower serum levels of NO in uremic patients. On the other hand, some studies found increased levels of endogen inhibitors of NO synthase (NOS) such as asymmetric dimethyl-arginine (ADMA). It is well known that higher levels of ADMA are present in dialyzed patients as well. Secretion of pro-inflammatory mediator’s platelet-derived growth factor and transforming growth factor can be caused by reduction in renal mass. Listed mediators are very potent inhibitors of NOS.[27],[28],[29]
Experimental studies on animals have shown that a decrease in renal mass leads to an increased synthesis of a potent vasoconstrictor, ET-1, which decreases the production of NO.[18],[25] In our study, serum levels of NO were significantly higher in CAPD patients, especially in patients with long-term CAPD. Potential explanation for these results can be found in increased production of NO from mesothelial cell. Davenport et al[30],[31] state that mesothelial and endothelial cells originate from the same germ layers. Besides this way of NO production, tissue macrophages, which are involved in inflammatory processes in peritonitis, represent a significant source of NO.[32] Patients treated with long-term CAPD develop peritonitis more often.[33] It is well known that long-term CAPD patients with confirmed hypotension have similar stroke volumes and heart rates as normotensive CAPD patients but with lower peripheral vascular resistance.[34] One of the possible explanations for this is that long-term CAPD patients start to produce vasodilators intensively, primarily NO and adrenomedullin which characterizes the progression of ESRD.
The main limitation of this study is that it is a single-center study with small number of patients.
From this study, it can be concluded that long term renal replacement therapy (CAPD) has a significant effect on overproduction of ET-1 and NO. This imbalance can lead to disturbance in local blood flow control, and these pathophysiological mechanisms can cause significant hemodynamic disturbances that may lead to hypertension and atherosclerosis. Consequently, complications of the same process which have irreversible character occur, which might explain the worsening of cardiovascular function and premature deaths.
Conflict of interest: None declared.
References 
Correspondence Address:
Pedja Kovacevic
Medical Intensive Care Unit, University Clinical Centre of the Republic of Srpska
Bosnia and Herzegovina
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/1319-2442.367823
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