Objectives: We aimed to evaluate the relationship between interleukin-8 and the oxidant-antioxidant system in end-stage renal failure patients with and without diabetes mellitus undergoing regular hemodialysis treatment.
Materials and Methods: Plasma levels of malondialdehyde and whole blood reduced glutathione were measured as markers of the oxidant and antioxidant systems, respectively. Plasma interleukin-8 levels were measured by enzyme-linked immunosorbent assay.
Results: When compared with controls, plasma interleukin-8 levels were elevated in both diabetic and nondiabetic end-stage renal disease patients. Plasma malondialdehyde levels were statistically significantly higher in end-stage renal disease patients with and without diabetes mellitus than they were in controls; however, reduced glutathione levels were statistically significantly lower in diabetic and nondiabetic end-stage renal disease patients than they were controls.
Conclusions: In end-stage renal disease patients with and without diabetes mellitus, elevated interleukin-8 levels and decreased reduced glutathione levels may be attributed to increased oxidative stress due to inflammation. In other words, increased reactive oxygen species may induce interleukin-8 production and result in diminished reduced glutathione levels. Our data suggest a relationship between interleukin-8 and the oxidant-antioxidant system in end-stage renal failure patients.
Key words : Cytokine, Malondialdehyde, Glutathione
Since 1997, accumulating experimental evidence has shown that reactive oxygen species (ROS) play a key role in the pathophysiological pathways of a wide variety of clinical and experimental renal diseases [1-3]. These ROS, including the superoxide anion, the hydroxyl radical, hypochlorous acid, and peroxynitrite may be generated by activated neutrophils, monocytes, and mesangial cells during metabolic processes . ROS have been shown to be primary mediators in glomerulonephritis, and they are factors in the regulation of glomerular permeability to proteins, development of morphologic lesions, and alteration of glomerular hemodynamics (ie, reductions of glomerular blood flow and glomerular filtration rate) . ROS, generated either extracellularly or intracellularly through a ligand-receptor interaction, can function as signal transduction molecules to activate chemotactic cytokine interleukin-8 (IL-8) .
IL-8 is an important cytokine in the process of inflammation. It is produced by a wide variety of cell types including monocytes, fibroblasts, endothelial cells, keratinocytes, and Langerhans cells in response to inflammatory stimuli . Endothelial-derived IL-8 secreted into the subendothelial matrix or bound to the surface of the endothelium promotes neutrophil adherence and migration [8, 9].
It has been suggested that antioxidants inhibit production of IL-8 . Glutathione (GSH) is an important antioxidant that eliminates toxic peroxides and aldehydes from the cell and indirectly maintains vitamins C and E in their reduced and functional forms in tissue [11-13]. In renal diseases, there is profound imbalance between the oxidant and the antioxidant systems . In addition, elevated oxidative stress induces the synthesis of chemokines .
In this study, we evaluated the relationship between IL-8 and the oxidant-antioxidant system in patients with end-stage renal disease (ESRD) with and without diabetes mellitus (DM) undergoing regular hemodialysis treatment.
Materials and Methods
The study population included 71 ESRD patients with DM (21 with type 1 DM and 42 with type 2 DM) and without DM (4 with chronic glomerulonephritis, 3 with chronic pyelonephritis, and 1 with Alport’s syndrome) undergoing regular hemodialysis and 94 healthy subjects who served as controls. Prior to the study, the study protocol was approved by our local institutional ethics committee. The protocol conforms with the ethical guidelines of the 1975 Helsinki Declaration. Written informed consent was obtained from all of the subjects.
Diabetic patients were divided into type 1 and type 2 to observe whether diabetes type had any effect on the parameters we measured. ESRD patients were recruited from various hemodialysis centers. Whole blood with ethylenediaminetetraacetic acid (EDTA) was obtained from the Istanbul Medical Faculty, the Marmara University School of Medicine, Okmeydani State Hospital, and Baskent University in Istanbul, Turkey. Control subjects were selected among people without a history of renal diseases and without DM in their first-degree relatives. Patients with ESRD with creatinine clearances of less than 15 mL/min were included in the study.
Blood samples (10 mL) were taken just before the dialysis procedure, and whole blood reduced GSH levels were studied on the same day. Antioxidant status was determined by measuring reduced GSH levels in whole blood according to the method of Beutler and associates . After whole blood was hemolyzed with distilled water, reduced GSH levels were determined using a 5,5’dithio-bis-(2-nitrobenzoic acid) reagent. The formed colored complex was measured at 412 nm by a spectrophotometer. The remainder of the blood was used to obtain plasma to investigate IL-8 and malondialdehyde (MDA) levels. The extent of plasma lipid peroxidation was assessed by measuring MDA, the end product of lipid peroxidation, using a thiobarbituric acid assay according to the method of Yagi . After a reaction of thiobarbituric acid with MDA, the reaction product was extracted in butanol, and its absorbance was determined spectrophotometrically at 535 nm. Plasma IL-8 levels were determined with an enzyme-linked immunosorbent assay kit (Pelikine Compact, CLB, Amsterdam, Netherlands) according to the manufacturer’s instructions. The detection limit of the assay was 0.6 pg/mL.
Statistical analyses were performed using SPSS software (Statistical Package for the Social Sciences, version 10.0, SSPS Inc, Chicago, IL, USA). Clinical laboratory data are expressed as means ± SD. Mean values between patients and controls were compared using an unpaired t test. Values for P less than .05 were considered statistically significant.
The demographic characteristics of the study groups are given in Table 1. There were no significant differences with regard to age among diabetic ESRD, nondiabetic ESRD patients, and controls. Table 2 shows the biochemical parameters of the patient and the control groups. As expected, fasting plasma glucose levels were considerably higher in both type 1 and type 2 diabetic ESRD patients than they were nondiabetic ESRD patients and healthy controls (P < .01). When patients in the type 1 and type 2 diabetic groups were compared, no significant differences with regard to MDA, GSH, or IL-8 levels were found. Compared with controls, plasma IL-8 levels were elevated in both diabetic ESRD (P < .001) and nondiabetic ESRD patients (P < .01). Plasma MDA levels were statistically significantly higher in ESRD patients with and without DM than they were in controls (P < .01), but whole blood reduced GSH levels were significantly lower in diabetic and nondiabetic ESRD patients compared with controls (P < .01) (Table 2).
An expanding body of data now strongly suggests that chemokines contribute to inflammatory glomerular as well as tubulointerstitial diseases [18-20]. All types of renal cells can produce chemokines . ROS can upregulate chemokine expression and may represent a common mechanism of injury-induced chemokine generation .
In the present study, plasma IL-8 and MDA concentrations were markedly elevated in diabetic ESRD and nondiabetic ESRD patients compared with healthy controls. Increased MDA concentrations may be due to the elevated oxidative stress during inflammation in ESRD patients. Mezzano and associates  found that TNF-α, IL-8 inflammatory cytokines, and thiobarbituric acid-reactive substances, a marker of lipid peroxidation, were increased in patients with chronic renal failure compared with healthy controls. Oberg and associates  showed increased oxidative stress and acute-phase inflammation in patients with stage-3, stage-4, and stage-5 chronic kidney disease compared with healthy subjects. In our study, oxidative stress also may have induced IL-8 production by activating nuclear transcription factors in ESRD patients. Oxidative stress has been reported to mediate IL-8 synthesis . Recent studies have demonstrated that oxidative stress generated directly by exogenous H2O2 differentially induces IL-8 synthesis in epithelial and endothelial cells [25, 26]. IL-8 induction is associated with the activation of nuclear transcription factors, such as nuclear factor kappa B (NF-kB) and activator protein-1 in response to oxidative stress in various cell types [6, 27]. Since the IL-8 gene promoter region contains binding sites for activator protein-1 and NF-kB, activation of NF-kB leads to the production of mRNA species encoding IL-8.
GSH concentrations are closely correlated with the degree of renal failure. Our study showed that whole blood GSH levels were significantly lower in ESRD patients with and without DM compared with controls. It has been reported that increased ROS result in depletion of antioxidants . GSH is present in high concentrations in most prokaryotic and eukaryotic cells. Body fluids such as bile, glomerular filtrate, blood plasma, and epithelial cell lining also contain GSH. GSH can react with ROS in several ways. First, it can react as a reductant, reducing species such as H2O2 directly to water with the formation of oxidized glutathione (GSSG). Second, it can react directly with ROS, yielding GSSG . The GSH:GSSG ratio is therefore an indicator of oxidative stress. One limitation of our study is that we could not measure the concentration of GSSG. In our study, GSH depletion could have arisen from a decreased GSH:GSSG ratio. Oxidative stress might result in a decrease in this ratio because of elevated GSSG . GSH depletion also could be attributed to decreased GSH synthesis and/or increased GSH degradation. Oxidative stress or depletion of GSH and subsequent increases in cytosolic GSSG in response to oxidative stress lead to the activation of NF-kB . Depletion in GSH by itself could contribute to the progression of uremia because it has been demonstrated that GSH depletion in rats leads to acute renal failure [31, 32]. Some authors have suggested that a naturally occurring thiol antioxidant compound such as ergothioneine could inhibit transcriptional activation of IL-8 . Redox-sensitive transcription factors such as NF-kB and activator protein-1 (their activation is affected by the redox GSH balance) have been shown to be activated in epithelial and inflammatory cells during oxidative stress and inflammation, leading to the upregulation of several proinflammatory genes .
In conclusion, in ESRD patients with and without DM, elevated IL-8 and decreased GSH levels may be attributed to increased oxidative stress, which results from inflammation during ESRD. In other words, increased MDA may induce IL-8 production and also result in decreased GSH. Our data suggest a relationship between IL-8 and the oxidant-antioxidant status in ESRD patients.
Volume : 5
Issue : 1
Pages : 610 - 613
Departments of 1Neuroscience and 2Molecular Medicine, Istanbul University, Institute for Experimental Medicine; and 3Department of Nephrology, University of Marmara, Istanbul, Turkey
Address reprint requests to: Assoc Prof Makbule Aydin, Istanbul University, Institute for Experimental Medicine, Department of Neuroscience, Vakýf Gureba Cad. 34280, Capa, Istanbul, Turkey
Phone: 00 90 212 414 20 00 / 33356
Fax: 00 90 212 532 41 71
Table 1. Demographic characteristics of the subjects
Table 2. Biochemical parameters of subjects