N-acetyl-cysteine is associated to renal function improvement in patients with nephropathic cystinosis
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- Pache de Faria Guimaraes, L., Seguro, A.C., Shimizu, M.H.M. et al. Pediatr Nephrol (2014) 29: 1097. doi:10.1007/s00467-013-2705-3
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Nephropathic cystinosis is an autosomal recessive systemic severe disease characterized by intralysosomal cystine storage. Cysteamine is an essential component of treatment. There is solid evidence that cystine accumulation itself is not responsible for all abnormalities in cystinosis; there is also a deficiency of glutathione in the cytosol. Patients with cystinosis can be more susceptible to oxidative stress.
The patient cohort comprised 23 cystinosis patients (16 males) aged <18 years (mean age 8.0 ± 3.6 years) with chronic kidney disease class I–IV with good adherence to treatment, including cysteamine. Oxidative stress was evaluated based on the levels of serum thiobarbituric acid-reactive substances (TBARS), and renal function was evaluated based on serum creatinine and cystatin C levels and creatinine clearance (Schwartz formula). N-Acetylcysteine (NAC), an antioxidant drug was given to all patients for 3 months (T1) at 25 mg/kg/day divided in three doses per day. The measured values at just before the initiation of NAC treatment (T0) served as the control for each patient.
Median serum TBARS levels at T0 and T1 were 6.92 (range 3.3–29.0) and 1.7 (0.6–7.2) nmol/mL, respectively (p < 0.0001). In terms of renal function at T0 and T1, serum creatinine levels (1.1 ± 0.5 vs. 0.9 ± 0.5 mg/dL, respectively; p < 0.0001), creatinine clearance (69.7 ± 32.2 vs. T1 = 78.5 ± 33.9 mL/min/1.73 m2, respectively; p = 0.006), and cystatin c level (1.33 ± 0.53 vs. 1.15 ± 0.54 mg/l, respectively; p = 0.0057) were all significantly different at these two time points. Serum creatinine measurements at 6 (T −6) and 3 months (T −3) before NAC initiation and at 3 (T +3) and 6 months (T +6) after NAC had been withdrawn were also evaluated.
During the 3-month period that our 23 cystinosis patients were treated with NAC, oxidative stress was reduced and renal function significantly improved. No side-effects were detected. Larger and controlled studies are needed to confirm these findings.
KeywordsNephropathic cystinosisOxidative stressGlomerular filtration rateN-AcetylcysteineCysteamine
Nephropathic cystinosis is a severe autosomal recessive systemic disease that is characterized by intralysosomal cystine storage  due to a defect in cystinosin, a lysosomal cystine carrier, caused by a mutation in the CTNS gene . Patients present with Fanconi syndrome by the age of 6 months, and without specific treatment with cysteamine (a depleting-drug of cystine storage) , the glomerular filtration rate (GFR) progressively deteriorates towards end stage renal disease (ESRD) during approximately the first decade of life. Other organs, such as the eyes and thyroid, and, subsequently, the central nervous system, muscles, and pancreas are involved, among others .
Unfortunately, even when cysteamine treatment begins early, the patients usually develop ESRD by approximately their second or third decade of life, and they can also present extrarenal compromise [4–6]. Experimental evidence indicates that cystine accumulation itself is not responsible for all of the abnormalities in cystinosis, and several in vitro studies of cellular models have shown a link among a variety of factors, including cystine accumulation, altered adenosine trisphosphate (ATP) metabolism, increased apoptosis, and cell oxidation [7, 8]. Wilmer et al.  demonstrated increased levels of oxidized glutathione (GSSG) in cystinotic proximal tubular epithelial cell lines; similarly, Levtchenko at al. did not observe a decrease in the total intracellular reduced glutathione (GSH) content in cystinotic cells, although they did observe elevated GSSG levels in cystinotic cells, resulting in an increased GSSG/total GSH (%) ratio . Conversely, Vitvitsky et al. did not observe an abnormal redox status in cystinotic cells . Taken together, these findings may suggest that cystinosis patients can be more susceptible to oxidative stress, particularly under stressful conditions, and that this susceptibility can contribute to the progression of renal disease. In a previous study, we demonstrated increased levels of thiobarbituric acid-reactive substances (TBARS), which are markers of lipid peroxidation (oxidative stress), in the sera of cystinosis patients ; this observation suggests that the increased oxidative stress plays a role in the pathogenesis of the disease. This hypothesis is corroborated by the fact that this disease is accompanied by a deficiency of glutathione, the major intracellular antioxidant, in the cytosol.
N-Acetylcysteine (NAC) is an antioxidant drug; its potential to decrease oxidative stress has been already demonstrated in several situations, such as with ifosfamide toxicity , cyclosporine toxicity , and cisplatin nephrotoxicity , as well as in oxidant-stress-mediated renal tubular injuries in patients at high risk of contrast nephropathy .
The aim of this study was to evaluate the effect of NAC on the renal functions of cystinosis patients treated with cysteamine and to detect the side-effects.
Patients and methods
Male and female cystinosis patients with chronic kidney disease classes I–IV  and younger than 18 years who showed good adherence to treatment, including cysteamine, were enrolled in the study. The diagnosis was based on clinical findings, Fanconi syndrome and results of the ophthalmologic exams. During the study, all patients were under metabolic and hydro electrolyte control, i.e., they were clinically stable, and the results of laboratory tests fell within the normal range or close to it.
The renal function evaluation was based on measurements of serum cystatin C and creatinine levels, and the creatinine clearance was estimated using the Schwartz formula . We chose the Schwartz formula because the 24-h urine collection is difficult to accomplish in polyuric patients. Cystatin C and serum creatinine were measured using the nephelometric method and a modified Jaffe’s method, respectively.
Oxidative stress was evaluated using the TBARS assay . Briefly, 0.2 ml of serum was diluted in 0.8 ml of distilled water, followed immediately by the addition of 1 ml of 17.5 % trichloroacetic acid. Thereafter, 1 ml of 0.6 % thiobarbituric acid, pH 2, was added to the sample which was then placed in a boiling water bath for 15 min, after which it was allowed to cool. Subsequently, 1 ml of 70 % trichloroacetic acid was added, and the mixture was incubated for 20 min, then centrifuged for 15 min at 2,000 rpm. The optical density of the supernatant was read on a spectrophotometer at 534 nm against a reagent blank. The quantity of TBARS was calculated using a molar extinction coefficient of 1.56 × 105 M –1 cm –1. The normal range is 1.60 ± 0.04 nmol/ml .
The levels of the hepatic enzymes alanine transaminase and aspartate transaminase levels were determined to detect possible hepatotoxicity by NAC. We also asked each patient if they had complaints.
The intraleukocyte cystine content was measured by high-performance liquid chromatography at the UCSD Biochemical Genetics Laboratory, San Diego, California, USA, to verify whether the cysteamine dosage used was effective in reducing the intracellular cystine content and also to check treatment adherence.
In this type of study, it is difficult to have a control group because cystinosis is a rare disease. Therefore, we considered each patient as his/her own control.
We employed a 25 mg/kg/day dosage of NAC, which was divided into three doses and administered to all patients; after 3 months of NAC treatment, we observed the results. This dosage was chosen based on a previous study of renal transplant adult patients who demonstrated the benefits of NAC in preserving renal function at a dosage of 600 mg twice daily without side-effects ; based on dosage for an adult patient weighing 50 kg, we considered that 25 mg/kg/day could be employed in children at a maximum dosage of approximately 1200 mg/day.
Time points of the study
The time points used in this study were T-6 and T-3, 6 and 3 months, respectively, before initiation of NAC treatment; T0, the time immediately prior to the initiation of NAC treatment; T1, 3 months of NAC use; T +3 and T +6, 3 and 6 months, respectively after NAC treatment had been withdrawn.
The following variables were measured at T0 and T1: serum creatinine, creatinine clearance using the Schwartz formula, serum cystatin C, serum TBARS levels, and hepatic enzymes. To compare the results, we also evaluated the serum creatinine and creatinine clearance data at T −3 and T −6 and at T +3 and T +6. We did not measure the cystatin C levels after NAC treatment was withdrawn.
Homogenous data are expressed as the mean ± standard deviation, and non-homogenous data are expressed as the median and range. A paired t test was employed to compare the variables with a normal distribution, and the Wilcoxon test was used to compare the variables without normal distribution. To evaluate all times of serum creatinine collection before, during, and after the NAC had been withdrawn, we used an analysis of variance. p < 0.05 indicated a statistically significant difference.
The study was approved by the local ethics committee, and informed consent was obtained from the parents of all patients before they were included in the study. The study is registered in clinicaltrials.gov under the identification number NCT01614431.
Patient’s clinical data
Total number of patients
Gender, male/female (n)
Age at cystinosis diagnosis (months)
Age at initiation of cysteamine treatment (months)
Age at initiation of N-acetylcysteine (NAC) treatment (months)
94 ± 38
Serum TBARS and renal function measurements
To better compare the renal function prior to NAC administration and after NAC withdrawal, Figs. 2 and 3 include measurement data obtained at 3 and 6 months prior to initiating NAC treatment (T −3 and T −6, respectively) and at 3 (T +3) and 6 months (T +6) after NAC treatment had been withdrawn. The serum creatinine level (in mg/dl) was 0.88 ± 0.50 at T −6, 0.93 ± 0.55 at T −3, 1.10 ± 0.50 at T0, 0.90 ± 0.50 at T1, 1.17 ± 0.50 at T +3, and 1.34 ± 0.50 at T +6 (Fig. 2). Creatinine clearance (in mL/min/1.73 m2BS) was 82.3 ± 40.6 at T −6, 80.6 ± 38.9 at T −3, 69.7 ± 32.2 at T0, 78.5 ± 33.9 at T1, 59.1 ± 18.8 at T +3, and 52.1 ± 16.3 at T +6.
The levels of hepatic enzymes were normal before the NAC treatment and remained normal.
No side-effects were observed.
Nephropathic cystinosis is a severe disease. Although cysteamine treatment improves its prognosis, it does not completely abolish renal failure. Gahl et al.  emphasized that “cysteamine is not 100 % effective” even after taking all of the appropriate steps because some patients present extrarenal complications and renal failure at the age of 20 years. Likewise, Greco et al.  also demonstrated that ESRD cannot be prevented in most patients but that it is only postponed to the second or third decade of life; late-onset complications ultimately develop in a substantial proportion of patients. In addition, cysteamine compliance is challenging because of inconvenient dosing requirements and barely tolerable side-effects . Ergo, nephropathic cystinosis is still challenging for treating physicians.
New treatments have been explored for cystinosis [21, 22], but currently the most important objective is to preserve renal function and avoid extrarenal compromise. In this sense, knowledge about the disease pathogenesis can help to improve interventions. Several recent studies have demonstrated altered ATP metabolism and increased apoptosis and cell oxidation in cystinosis . Increased autophagy is involved and leads to increased reactive oxygen species (ROS) production .
In a previous study by our group, we compared serum TBARS levels in healthy children who had been matched with cystinosis patients and detected a significant difference, with increased levels of serum TBARS in the cystinosis patients. This abnormality was not correlated with the degree of renal insufficiency. .
Interestingly, it has been recently shown that cysteamine, aside from its cystine-depleting benefits, can increase intracellular GSH levels and restore cellular redox status, a mechanism which has been previously hypothesized to be responsible for the preservation of the kidney function of cystinosis patients treated with this drug . This mechanism further points to the possibility of using GSH precursors to preserve renal function in cystinosis patients. However, the mechanisms by which GSH restoration in cystinotic cells promotes the preservation of kidney function must still be determined.
Cysteamine is a potent antioxidant, and we hypothesized that by the addition of another anti-oxidant drug to the treatment regimen, such as NAC, might potentially decrease oxidative stress and preserve renal function. NAC is generally employed as a mucolytic agent. It has a thiol group that is a source of L-cysteine and reduced glutathione and serves as an antioxidant by virtue of its interaction with ROS . There are several reports of the benefits of NAC in decreasing oxidative stress in different situations; most of these studies evaluated serum levels of TBARS [13–16, 20, 24, 25].
Therefore, we decided to add NAC (at a dosage of 25 mg/kg/day) to the treatment regimen of cystinosis patients already receiving cysteamine. We observed that the level of serum TBARS fell and that renal function markers, serum cystatin C and creatinine levels, and creatinine clearance all improved. We also observed that 3 and 6 months after NAC had been withdrawn, there was a decline in the renal function status, as measured by serum creatinine and creatinine clearance. We believe this result was due to disease evolution.
Cystatin C was not measured after NAC was withdrawn. However, in a previous study by our group, we analyzed 103 blood samples from 26 patients and found a significant statistical correlation between serum creatinine and cystatin C levels (r = 0.81, p < 0.0001), between cystatin C level and creatinine clearance, as estimated by the Schwartz formula (r = −0.84, p < 0.0001), and between serum creatinine level and creatinine clearance (r = −0.97, p < 0.0001). We concluded that the expensive measurement of cystatin C had no advantages over the use of the other GFR markers .
The serum level of TBARS has been considered to be a good marker of oxidative stress in previous studies [16, 20, 24]. Therefore, in this study, we considered this method to be reliable for evaluating oxidative stress. Although, theoretically, this assay mainly measures malondialdehyde, it also measures other aldehydes that can react with TBA. It is important to keep this fact in mind when our results are being interpreted.
In conclusion, over a 3-month period, we used cysteamine and NAC in cystinosis patients without renal replacement therapy and observed reduced oxidative stress and significantly improved renal function. In addition, no side-effects were observed. Our results, although derived from a small, uncontrolled study, might indicate that the addition of NAC to conventional treatment with cysteamine may have some transient beneficial effect on the renal function of cystinosis patients. The absence of long-term follow-up data on the efficiency and toxicity of NAC precludes its routine use in cystinosis patients and warrants further study.
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) process number 2010/10622-0.