Balkan endemic nephropathy (BEN) represents a chronic tubulointerstitial nephropathy which is followed by the progression of kidney fibrosis to end-stage kidney failure. The critical involvement of poisons in food (aristolochic acid (AA), ochratoxin, and heavy metals) and selenium deficiency are among nutritive factors which contribute to the pathogenesis of BEN, due to reactive oxygen species (ROS) liberation and/or decreased antioxidative defence system. The aim of the study is to distinguish a possible systemic and local origin of ROS through the measurement of xanthine oxidase (XO) activity in urine and plasma, along with the determination of the oxidative changes in lipids and proteins. The study included 50 patients with BEN and 38 control healthy subjects. We noted increased levels of both thiobarbituric acid-reactive substances (TBARS) and advanced oxidation protein products (AOPPs) in the plasma of patients with BEN, compared to the control group (
Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy characterized by an unpredictable onset and a gradual progression to end-stage renal disease. Upper urothelial cancer (UUC) of the pelvis and ureter has increased prevalence in BEN patients [
The pathophysiology of BEN has been elaborated in a number of reports [
Increased oxidative stress (OS) caused by overproduction of reactive oxygen species (ROS) and deficiency of antioxidative defence systems may be possible common mechanisms for all of the factors mentioned above, including the progressive renal injury of the disease [
One of the hypothetical mechanisms for ROS production in BEN may be the xanthine oxidase (XO) reaction. XO is the oxidative radical-forming isoform of xanthine oxidoreductase. XO is the main enzyme involved in uric acid production, acting as the final metabolite of the adenine nucleotides. Simultaneously with the production of uric acid, XO activity liberates hydrogen peroxide and superoxide anion, well-established prooxidant molecules [
The aim of the present study is to determine whether OS is involved in the pathogenesis of BEN, observed via oxidative changes in lipids and proteins in plasma and urine.
All the reagents were purchased from Sigma (St. Louis, MO, USA). All chemicals used were of analytical grade.
We recruited patients with BEN from the Institute of Nephrology, Clinical Centre of Nis, Serbia, in accordance with a standard diagnostic protocol [
Baseline characteristics of BEN study cases and control group.
Characteristics | BEN | Controls | |
---|---|---|---|
Men | 30 (60%) | 22 (57.5%) | 0.866 |
Women | 20 (40%) | 16 (42.5%) | |
Age (years) | 72 (52.6-86.7) | 73 (65.05-83.95) | 0.377 |
SCr (mol/l) | 120.2 (70.22-606.4) | 80.2 (68.63-125.15) | |
CCr (ml/min) | 35.61 (7.93-87.41) | 65.9 (23.09-108.7) | |
UCr (mmol/l) | 7.02 (1.77-22.75) | 10.23 (5.2-23.53) | |
UPCr (mg/mmol) | 20.61 (5.43-423.47) | 10.42 (5.22-24.24) | |
UACr (mg/mmol) | 1.22 (0.18-60.72) | 0.87 (0.2-12.07) | |
Hgb (g/l) | 13.1 (8.7-131.6) | 122 (12.71-152.9) | |
Glucose (mmol/l) | 4.66 (3.8-6.4) | 5.3 (4.31-6.89) | |
94.81 (5.85-4754) | — | — | |
U protein (mg/l) | 215 (36.5-1510.5) | 107 (50-496) | |
U albumin (mg/l) | 16.48 (1.44-434.24) | 8.67 (2.14-234.65) | 0.197 |
Values are expressed as mean value (or percent) and median value (5th-95th percentiles). SCr: serum creatinine; CCr: creatinine clearance; UCr: urea : creatinine ratio; UPCr: urine protein : creatinine ratio; UACr: urine albumin : creatinine ratio; Hgb: hemoglobin.
We analysed plasma and urine for biochemical parameters on an A24 automatic analyser for in vitro diagnostics (Biosystems SA).
Lipid peroxidation in urine and plasma in terms of TBARS formation was determined using a slightly modified method of Nabavi et al. [
The concentration of AOPPs in plasma and urine was determined by spectrophotometric technique according to the method of Witko-Sarsat et al. [
The specific activity of XO was evaluated in plasma, spectrophotometrically, according to the liberation of uric acid by using xanthine as substrate, in the absence of NADH in cases in which only molecular oxygen was the acceptor of electrons. Uric acid was stoichiometrically formed from xanthine, and it was measured at 293 nm. XO activity was expressed in IU/l [
Quantitative variables were presented as arithmetic means with standard deviation (
The concentration of TBARS in plasma and urine is shown on Figure
TBARS values in patients with BEN and controls. Data are
AOPP values in BEN patients and controls. Data are
AOPP values in patients with BEN and controls: plasma/urine ratio.
Xanthine oxidase activity in patients with BEN and controls. Data are
XO values in patients with BEN and controls: plasma/urine.
BEN affects a large number of inhabitants of endemic areas. In the last decades, epidemiologic findings have revealed that BEN is an environmentally induced disease. Aristolochia, ochratoxin, selenium deficiency, and heavy metals are among the most important factors contributing to the onset and development of this disorder. However, the influence of these factors in association with chronic tubulointerstitial nephropathy and end-stage renal disease remains undetermined.
The association of food containing AA with BEN and UUC was initially documented in 1969 by Ivić [
Heavy metals are also implicated in the pathophysiology of BEN. Among them, increased levels of silica, lead, uranium, copper, cobalt, zinc, manganese, arsenic, titanium, barium, aluminium, chromium, strontium, cadmium, bismuth, molybdenum, nickel, tungsten, and antimony in water and soil have been documented in areas with increased incidence and prevalence of BEN [
The deoxyribonucleic acid DNA-AA adducts induced by AA produce a particular molecular signature in the kidneys with BEN nephropathy. However, the pathophysiological mechanism whereby AA results in renal injury is still unclear. Declèves et al. [
Among mycotoxins, OTA was documented to be an OS inducer [
The kidneys are extremely vulnerable to ROS damage, because of long-chain-polyunsaturated fatty acids present in cell membranes and higher local concentration of excretory toxic products. The results of our study demonstrated a higher plasma level of TBARS in patients with BEN compared to controls. One of the first events in oxidative cellular injury is the oxidization of membrane lipids. Lipid hydroperoxides are nonradical intermediates derived from unsaturated fatty acids, phospholipids, glycolipids, cholesterol esters, and cholesterol itself. Their formation occurs in an enzymatic or nonenzymatic reaction involving ROS. Measuring a breakdown product like TBARS, a nephrotoxic molecule which is a biological marker of OS, is the most frequently performed method for determination of lipid peroxidation [
Circulating AOPPs serve as a marker of protein oxidation status. Our study showed that AOPPs were significantly elevated both in the plasma and urine of patients with BEN. Li et al. [
ROS can directly damage the deoxyribonucleic acid of proximal tubular cells and accelerate programmed cell death. To support this hypothesis, it was reported in a previous study that AA depleted the antioxidant glutathione in human renal tubular cells (HK-2) leading to tubular cell death [
Another potential source of ROS could be uremic toxins in patients with CKD. A higher level of those toxins promotes systemic inflammation via priming polymorphonuclear leukocytes and stimulating CD-8+ cells [
Our hypothesis was that the source of free radicals could be XO activity, since the liver, gut, and kidney represent XO-rich organs. XO-induced ROS production is known to be elevated in septic development by significant liver and kidney injuries, and XO inhibitors are one of the protectors from kidney injuries [
All findings mentioned above led to the expectancy of a higher activity of XO in BEN patients since there were many causes which would raise its activity, but this was not the case. Our results showed a lower XO activity in patients with BEN when compared to healthy control subjects. Plasma/urine ratio showed even more significant differences, which may point to a systemic effect of the enzyme. There are only a few patients with BEN who suffered from gout. One of the possible reasons would be the lack of actual substrates for XO, like urine nucleotides. Lower kidney mass, accompanied with diminished functional tissue, may be responsible for a lower substrate level. Furthermore, studies showed the importance of AA in the formation of DNA-AA adducts [
The level of lipid peroxides and AOPPs is significantly increased in BEN patients compared to controls. We hypothesize that both local and systemic overproduction of ROS might play a pivotal role in the pathogenesis of BEN which leads to end-stage renal disease and even cancerogenesis. Our results may for the first time demonstrate that XO would not be considered a direct systemic or local contributor to ROS production in BEN, most probably because of the diminished kidney functional tissue mass and AA-induced changes in purine nucleotide conformation. A low XO activity might also prevent gout in patients with BEN.
The data used to support the findings of this study are included within the article.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This work was supported by the Ministry of Science and Technological Development, Republic of Serbia (Projects 43012, 31060, and 41018), and by the Serbian Academy of Sciences and Arts, a branch in Nis (Projects О-06-17 and О-07-17). This research was funded by the Medical University of Nis, by internal scientific project number 45.