Effectiveness of n-acetylcysteine for preserving residual renal function in patients undergoing maintenance hemodialysis: multicenter randomized clinical trial

Abstract

Objective.To investigate the efficacy and safety of oral n-acetylcysteine (NAC) for preserving residual renal function in patients undergoing hemodialysis.

Design, setting, and participants.Randomized, multi-center, parallel-group, open-label clinical trial (registration no.IRCT 2014071418482N1).Between August and December 2014, 54 patients who have been undergoing hemodialysis for at least three months and had residual urine volume >100ml/24h from three dialysis centers were randomly allocated to NAC or no medication.

Interventions.Effervescenttablets of NAC 2400mg daily (in two divided doses) vs. no NAC for three months.

Main outcome measures.Residual renal function evaluated by (1) estimated glomerular filtration rate (GFR), (2) 24h urine volume, and (3) renal Kt/V. GFR and Kt/V were determined at baseline and after three months. 24h urine volume was measured at baseline, after one, two, and three months.

Results.Intention-to-treat analysis was performed on 47 patients (NAC=26, control=21). GFR in patients receiving NAC improved, whereas in the control arm a decline of 1.0 mL/min/1.73 m2 was recorded (3.59 vs 21.11 mL/min/1.73 m2, effect size=17.0%, p=0.004). For 24h urine volume, the between group difference after one month was significant (669 vs 533 ml/24h, effect size=15.4%, p=0.004). After three months, 24h urine volume in the NAC arm was on average 137 ml higher than in the control group, and the difference reached near significance (673 vs 536 ml/24h, p=0.072). In the follow up visit, Kt/V was higher in the NAC arm but the difference did not reach statistical significance (0.81 vs 0.54, p=0.152). NAC was discontinued in three patients due to anaphylactoid reaction (n=1), and gastrointestinal discomfort (n=2). No serious adverse events were documented.

Conclusion.Three months treatment with NAC appears to be effective in preserving renal function in patients undergoing hemodialysis and the medication is generally well-tolerated.

Introduction

Residual renal function (RRF) is a consequential prognostic factor in patients with end-stage renal disease (ESRD). A breadth of evidence in the past two decades has shown that diminished RRF is a robust predictor of mortality and morbidity in ESRD patients undergoing dialysis(1-4). An analysis of the Netherlands cooperative study on the adequacy of dialysis (NECOSAD-2) demonstrated that in patients undergoing peritoneal dialysis (PD), each 1 mL/min/1.73 m2 increment in residual glomerular filtration rate (GFR), curtails mortality risk by 12%(5). In a two-year prospective study of patients undergoing hemodialysis (HD), a significantly increased mortality risk of 227% among patients with no RRF was documented; the observed association was independent of dialysis duration, or the presence of pre-existing cardio-metabolic risk factors (6). Furthermore, the rate of decline in RRF while on dialysis, has been suggested to be of prognostic significance. In a study of 270 PD patients, Liao et al stratified patients into slow, intermediate, and fast decliners based on the rate of RRF loss(7). Over an average follow-up period of 45 months, it was shown that rapid decline in RRF is independently associated with poorer survival and an increased risk of peritoneal-to-hemodialysis switch due to treatment technique failure(7).

Given the strong association betweendiminished RRF and poor outcome in dialysis patients, strategies aimed at maintaining the remaining renal function, or at least retarding its decline rate are of critical importance(8, 9). Recently, non-randomized, short-term, pilot studies have shown that oral administration of n-acetylcysteine (NAC), a thiol-containing antioxidant, might be beneficial in preserving RRF in ESRD patients undergoing HD (10, 11) and PD(12). It is hypothesized that NAC, through replenishing intracellular glutathione (GSH) reserve(13), an innate potent anti-oxidant system, ameliorates oxidative stress and its accrued cellular and vascular damage(14), ultimately protecting the kidneys from progressive loss of function.Further, it has been suggested that NAC might exert vasodilatory effects on renal vasculature through nitric oxide and prostaglandin-dependent pathways(15, 16).

These findings, while promising, have not yet been corroborated in randomized clinical trial settings. Therefore, in the present multi-center parallel-group, randomized clinical trial, we aimed to investigate the efficacy and safety of oral NAC in maintaining residual glomerular filtration rate, urine volume, and dialysis adequacy in a cohort of patients undergoing maintenance HD over a period of three months.

Patients and Methods

Patients

The present study was planned as an open-label, parallel-group, randomized, multi-center clinical trial. Between August and December 2014, patients receiving chronic HD in three dialysis centers affiliated with Tehran University of Medical Sciences (Tehran, Iran) were assessed for eligibility. Subjects were considered eligible if they met the following inclusion criteria: (1) had end stage renal disease (ESRD) and were undergoing regular HD three times a week for at least three months; and (2) had a residual 24-hour urine volume of equal to or greater than 100 ml. Patients with acute renal failure as the indication of dialysis were not included. Further, patients taking prescribed or over-the-counter anti-oxidants (e.g. herbal supplements, vitamin E and C, and NAC) were excluded. All procedures dealing with human subjects were conducted in accordance with the guidelines laid down in the latest revision of Helsinki declaration. Written informed consent was obtained from all participants prior to enrollment. Patients who did not agree to participate were not enrolled. Ethics committee of the School of Medicine approved the trial protocol. The trial is registered at the Iranian Registry of Clinical Trials (Registration No. IRCT 2014071418482N1), a primary registry in the World Health Organization’s International Clinical Trials Registry Platform (ICTRP).

Trial design and treatment allocation

Initially 158 patients were assessed for eligibility and 54 met the inclusion criteria. Using a simple randomization software in Microsoft Excel®, numbers 1-55 were generated in a fashion to be unsorted and unrepeated. With the random sequence available, numbers equal to or smaller than 27 were allocated to the NAC arm, and the rest were designated to the control group. Patients in the NAC arm received 1200 mg NAC twice daily (2400 mg in total, ACC 600 mg effervescent tablets, Hexal®, Germany).The routine protocol of treatment including anti-hypertensive medications, anti-hyperlipidemic medications, anti-hyperglycemic medications (in patients with diabetes), and aspirin (if indicated) were continued as before. Patients in the control group received no anti-oxidants and their standard treatment was continued as before.

Assessment

Upon enrollment, a detailed medical history was obtained from each participant and was recorded in a pre-designed questionnaire. Physical examination was then performed by the interviewing physician. After a dialysis session, weight was measured using a calibrated digital scale and the reading was recorded with 0.1 kg precision. Height was measured using a wall-mounted stadiometer and was recorded to the nearest 0.1 cm. Body surface area (BSA) was then calculated using the Gehan and George formula (1970). After at least 10 minutes of resting, blood pressure was measured in a sitting position using a standard mercury sphygmomanometer from both arms. The average of readings from left and right arms was recorded.

All laboratory samples were collected mid-week in the baseline and follow-up assessments. After collection of 24-hour urine samples,patients were also instructed to go on an overnight fasting of eight hours and the following day, 10 ml of venous blood was drawn and immediately sent to the laboratory for biochemical analysis. Serum and urine creatinine concentrations were determined using the Jaffe method. No calibration was done for creatinine values. Albumin and urea concentrations were measured by photometric methods using available commercial kits (ParsAzmun, Karaj, Iran). With serum/urine creatinine and urea concentrations available, 24-hour creatinine and urea clearance were determined and normalized to 1.73 m2 BSA. Glomerular filtration rate (GFR) was defined as the arithmetic mean of creatinine and urea clearance over 24 hours. To assess dialysis adequacy dialysis and renal Kt/V were calculated. Dialysis Kt/V was calculated using the 1993 Daugirdas equation(17). Dialysis adequacy was defined as a dialysis Kt/V of at least 1.2. Renal Kt/V was calculated using the formula presented by the NECOSAD-2 study group (2).

Outcome

Primary outcome measures were (1) change in GFR, (2) change in 24-hour urine volume, and (3) change in Kt/V after three months. GFR and Kt/V were measured at baseline and after three months using the methods described above. 24-hour urine volume was measured at baseline, after one, two, and three months (four measurements in total). In the monthly visits, patients’ adherence to medication, experience of possible side effects, and where indicated, reason for medication discontinuation was questioned and recorded. Drug therapy compliance was assessed by counting tablets left in the container at the end of each monthly visit and patients were considered compliant if at least 80% of all doses were taken.

Statistical analysis

Statistical analyses were done using the Statistical Package for Social Sciences (SPSS) version 20 (IBM Corp., Armonk, New York). Continuous variables with normal distributions are presented as mean ± standard deviation. Variables with a non-normal distribution are described as median (interquartile range). Categorical variables are presented as proportions. Independent t-test or Mann-Whitney U test (for non-normal variables) were employed to compare continuous variables between the trial arms. Distribution of categorical variables across trial arms was assessed using Chi square (or Fisher’s exact test where necessary). Univariate Analysis of Co-Variance (ANCOVA) was used to investigate the three-month difference in outcome variables between NAC and control arms. In each model, outcome variable of interest measured after three months was introduced into the model as the dependent variable. Intervention was entered as the independent variable. Outcome variable measured at baseline was placed into the model as a covariate to adjust for the possible between group differences at baseline. Effect size was calculated from partial eta squared. Based on Cohen’s recommendations, partial eta squared values of 1%, 6%, and 13% indicate small, medium, and large effect sizes, respectively. In all analyses, a p value of less than 0.05 was considered necessary to reject the null hypothesis.

Results

The flow diagram depicting enrollment, treatment allocation, and follow-up of the patients is presented in Figure 1. Fifty-four HD patients were enrolled and randomly allocated to NAC (n=27) or control (n=27) arms of the trial. In the NAC arm, one patient was lost to follow-up and the data for 26 patients were available for the final analysis. In the control arm, one patient moved and was transferred to another dialysis facility. Another two patients underwent kidney transplantation during the study period and were excluded. Three patients deceased and the cause of death was coronary heart disease in all three. Therefore, data for a total of 21 patients from the control arm were available for the final analysis.

Baseline characteristics of trial participants are presented in Table 1. The average age of the patients was 58 years and ranged from 27 to 85. Sixty-three percent of the patients were male and the female:male ratio was 1:1.7. The most frequent etiology of ESRD was hypertension (55.5%), followed by diabetes (35.2%). In 13% of the participants, no underlying cause for ESRD was identifiable. Patients were on HD for an average of four hours in each session. Weekly dialysis adequacy was achieved in 51.9% of the patients.Baseline characteristics of HD patients by trial arm who were included in the final analyzed sample are presented in Table 2. Age, sex, systolic and diastolic blood pressures, hematocrit, serum albumin, and ultrafiltration rate were comparable between the two groups (p>0.05 for all tests). Baseline GFR in the NAC arm was higher than the control arm (median: 2.77 vs. 1.70), but the difference did not reach statistical significance. Furthermore, baseline levels of 24-hour urine volume and also renal Kt/V were not significantly different between the trial arms (p=0.198 and 0.462), respectively. Dialysis adequacy was observed in 61.0% and 67.0% of the patients in NAC and control groups, respectively (p=0.768).

The comparative efficacy of NAC vsno antioxidant on improving indices of residual renal function and also renal Kt/V are presented in Table 3. After three months, GFR in the arm of patients receiving NAC improved, whereas in the control arm a decline of 1 mL/min/1.73 m2 was recorded. The difference between the two arms was significant (F=9.02, p=0.004), suggesting NAC treatment is effective in preserving renal function over three months. The changes in 24-urine volume between the trial arms after one, two, and three months are illustrated in Figure 2. The between group difference after one month was statistically significant (F=8.93, p=0.004, effect size=15.4%). 24-hour urine volume in the NAC treated patients was on average 137 ml higher than the control group, and the difference reached near significance (p=0.072, Table 3 and Figure 2). In the follow-up visit, the level of renal Kt/V was comparable between trial arms and NAC treatment had no effect on Kt/V improvement (p=0.152).

Three patients in the NAC arm discontinued treatment. One patient developed transient urticariaand pruritus one week after the initiation of treatment and the medication was withheld. The symptoms resolved spontaneously in a few days and oral antihistamines were prescribed as needed. Two patients reported gastrointestinal discomfort following NAC intake and requested to be withdrawn from the trial; medication was discontinued on that account.

Discussion

In the present randomized clinical trial, three-month effects of oral NAC on preserving renal function in a group of patients on chronic HD were investigated. While in the group of patients receiving no treatment residual GFR declined, GFR in patients receiving NAC was relatively sustained and even slightly improved. The between group difference was statistically significant and a large effect size of 17.0% for NAC treatment was documented. With regard to urine volume, NAC was superior to no treatment in improving 24-hour urine volume after one month and 24-hour urine volume was relatively sustained thereafter, suggesting that the beneficial effects of NAC on urine volume appear early in the treatment process and tend to be maintained over longer periods.

In concert with our findings, in a prospective study of ten patients undergoing PD, Feldman et al (2011) showed that four weeks treatment with 1200 mg NAC twice daily results in significant improvements in 24-hour urine volume (633 vs. 925 ml) and renal Kt/V (0.56 vs. 0.75)(12). NAC treatment also increased residual GFR (4.96 vs. 5.95)(12). A 2012 study by the same research group indicated that NAC treatment might also be beneficial in HD patients(10). In this subsequent study, 20 patients received NAC 1200 mg two times daily for two weeks; significant improvements in GFR (1.6 vs. 2.4), 24-hour urine volume (320 vs. 430), and dialysis adequacy as measured by Kt/V (0.19 vs. 0.29) were observed(10). Contrary to these observations, NAC treatment did not cause an augmentation of dialysis adequacy herein. It should be noted however, that in the study by Feldman et al, baseline renalKt/V values were markedly lower(11), indicating a sub-optimal dialysis adequacy in the majority of patients. It could be argued that NAC administration will benefit renal Kt/V when less than optimal conditions are present, and the effect attenuates in patients with better dialysis efficiency.

Our findingscomplement the previous reports of short-term NAC administration in improving RRF in HD patients (10) by showing administration of oral NAC over a longer period maintains RRF and is relatively safe. In the NAC arm of the trial and over the period of three months, no serious adverse events occurred. Adverse reactions associated with NAC are relatively uncommon and depend on the route of administration. With intravenous administration, anaphylactoid reactions (e.g. pruritus, flushing, bronchospasms, hypotension, and true anaphylaxis) are more common, whereas gastrointestinal discomfort (e.g. nausea, vomiting, and abdominal pain) appear to be more frequent when the medication is ingested orally(18). In the present study, gastrointestinal symptoms were reported in two patients (7.4%), and mild anaphylactoid reactions of pruritus and itching occurred in one patient (3.7%) which was readily controlled with discontinuation of the medication. It should be noted that most studies concerning the adverse reactions to oral and intravenous acetylcysteine have been conducted in the setting of acetaminophen poisoning where significantly higher doses are administered to achieve higher plasma concentrations(19). When NAC is used in lower doses, the frequency of adverse reactions is considerably low and the medication is well-tolerated(20). A meta-analysis of eight randomized clinical trials of oral NAC (400-1800 mg per day) for chronic bronchopulmonary disease including more than 2000 patients reported no serious adverse reactions requiring treatment in the studied population (21).

Patients with ESRD are subject to increased oxidative stress and its ensued cardiovascular complications(22). Reactive oxygen species and free radicals appear to be elevated due to the accumulation of uremic toxins(14, 23). Aside from the disease process itself, co-morbid and underlying conditions such as diabetes, hypertension, and dyslipidemia which are present in a sizable proportion of HD patients, also contribute to the status of enhanced oxidative stress(24). On the other hand, in ESRD patients,natural antioxidant defense systems are severely impaired and antioxidant capacity is diminished, further exacerbating the detrimental processes by which reactive oxygen species induce cellular damage(25). Theoretically, antioxidant treatment might have a putative role in avertingthe deleterious effects of oxidative stress, thereby improving clinical outcomes in HD patients. Yet, clinical trials investigating the efficacy of various antioxidants in HD patients have yielded conflicting results and the clinical benefits seem equivocal(23). In this regard, NAC is deemed to be an exception. NAC has been consistently shown to decrease markers of oxidative stress and improve clinical outcomes in observational and randomized clinical trials(26-29). In a placebo-controlled clinical trial, oral NAC 1200 mg daily significantly reduced cardiovascular, cerebrovascular, and peripheral vascular events in HD patients(29).