Balkan endemic nephropathy (BEN) represents a chronic tubulointerstitial nephropathy, followed by the progression of kidney fibrosis to the end-stage kidney failure. The critical involvement of food poisons (aristolochic acid, ochratoxin and heavy metals), from one side, and selenium deficiency from the other, are among nutritive factors that contribute to the pathogenesis of BEN, because of the reactive oxygen species (ROS) liberation and/or decreased anti-oxidative defence system. Taking into consideration renal failure and dramatically reduced kidney mass in BEN, the aim of the study was to distinguish possible systemic and local origin of ROS through the measurement of xanthine oxidase activity (XO) in urine and plasma, together with the determination of the oxidative changes in lipids and proteins. The study included 50 patients with BEN and 38 control healthy subjects. There were increased levels of both, TBARS and AOPP in the plasma of patients with BEN, compared to control (p < 0.001). The urinary level of AOPP was higher in patients with BEN when compared to control (p < 0.001). The specific activity of XO was significantly lower in plasma and urine of BEN samples, compared to control ones (p < 0.005). Taken together, our results may show for the first time that XO would not be considered as a direct systemic or local contributor to ROS production in BEN, most probably because of the diminished kidney functional tissue mass and/or AA-induced changes in purine nucleotide conformation. The increased AOPP and TBARS level in both plasma and urine in BEN may predict to ROS systemic liberation with toxic local effects.
Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy characterized by a treacherous starting of the disease and step-by-step progression to the end-stage renal disease. The appearance of the upper urothelial cancer (UUC) of the pelvis and ureter is more frequent than in other population
The concentrations and therefore the extent of Selenium deficiency is documented in rocks, soil, water, foodstuffs, and blood samples collected from endemic and non-endemic regions of BEN in the geographical area of Serbia (22-24). Some findings led to the conclusion that heavy metals and metalloids may also be the causes of BEN (25).
As a possible common mechanism for all the above mentioned factors may be increased oxidative stress (OS) caused by higher production of reactive oxygen species (ROS) and a deficiency in anti-oxidative defence system. In the pathologic process of progressive renal injury, reactive oxygen species (ROS) could play an important role. Reports say the influence of OS is emerging as a may be crucial reason for the high cardiovascular disease incidence among chronic kidney disease (CKD) patients. ROS are indeed extremely related to kidney dysfunction (26) and accumulated ROS is closely associated with high cardiovascular disease incidence (27). Also, all tubulointerstitial injuries are characterized by the generation of free oxygen radicals (28) highly nephrotoxic and very reactive metabolites released throughout the oxidative burst. As a consequence of this disbalance, it increases lipid per-oxidation, as the most significant mechanism of the cell membrane injury.
Proteins are also one substrate for ROS- induced injury. Recent studies found that plasma concentration of advanced oxidation protein products (AOPP) significantly raised with the progression of nephritic pathology in patients with a variety of CKD (29), likewise as in patients with diabetes (30).
Whether the mechanism of the ROS stimulated liberation is inflammation or decreased anti-oxidative defence, it mediates urinary tract injury by direct cytotoxic effects on proximal tubular cells, through altered renovascular responses, or even as an orchestrating event of many factors.
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 (26). Simultaneously with the production of uric acid, XO activity liberates hydrogen peroxide and superoxide anion, that are one among the main ROS and oxidative stress-inducers. There are plenty of pathological conditions during which increased plasma XO exist, like cholecystitis, shock, ischaemia-reperfusion injury, acute virus infection, adult respiratory distress syndrome, carcinogenesis (28). The question was whether XO activity may be one of the potential mechanisms involved in the pathogenesis of BEN-associated kidney injury and cancerogenesis of the upper urothelial tract.
The aim of the present study was to determine if the oxidative stress phenomena may be involved in the pathogenesis of patients with BEN, determined via oxidative changes in lipids and proteins in plasma and urine of patients with BEN.
All The reagents were purchased from Sigma (St. Louis, MO, USA) and were of the best business quality obtainable. All used chemicals were of analytical grade.
We recruited patients with BEN from the Institute of nephrology, Clinical Center of Nis, Serbia, based on a standard diagnostic protocol (31). Compared to the tubulointerstitial character of the disease, the clinical-biochemical diagnostic set used: 1) a history of the place where the patient was born and currently lives; 2) laboratory urine analysis: glomerular filtration (GFR) reduction; micro-albuminuria (proteinuria <1 g / 24h) associated with urinary markers tubular lesions (ß2-micro-globulin in urine), laboratory blood test: urea and creatinine; 3) radiological findings; 4) ultrasound of the kidney. The glomerular filtration rate defined the degree of diminished glomerular function (eGFR), which was less than 60 ml/min / 1.73. The estimates included in the study (50) were selected from the endemic areas around South Morava, with 38 healthy subjects comprised the control group. We showed demographic characteristics of BEN patient and control group on Table 1. Control patients did not have any acute or chronic illness or high blood pressure. Respondents were matched for gender and age.
Blood and urine patients with BEN and control groups were collected , centrifuged to separate plasma at 3000 rpm and stored at -20 oC until analysis.
We analysed plasma and urine for biochemical parameters on A24 Automatic analyser for In-Vitro diagnostics (Biosystems SA).
Lipid peroxidation product
Lipid peroxidation in urine and plasma in terms of TBARS formation was determined using a slightly modified method of Nabavi et al. (32). The quantity of MDA- reactive lipid per-oxidation products was measured at 532 nm against a blank. Concentration was expressed in µmol/L.
AOPP concentration
The concentration of AOPP in plasma and urine was determined by spectrophotometric technique according to the method of Vitko et al (29). AOPP concentration was expressed in μmol /L chloramine T.
XO activity
The specific activity of XO was evaluated spectrophotometrically, by using xanthine as substrate where uric acid formation was measured at 293 nm. XO activity was expressed in IJ/L. (33)
The results were expressed as mean ± SD. A p-value< 0.05 was accepted as statistically significant. We performed statistical analysis with Statistical Package for the Social Sciences for Windows (SPSS, version 11.0, Chicago, Illinois, USA).
Characteristics | BEN | Controls | P value |
Men | 31 (59.6%) | 22 (57.5%) | 0.866 |
Women | 21 (40.4%) | 16 (42.5%) | |
Age (years) | 73 (53.6-86.7) | 73 (65.05-83.95) | 0.377 |
SCr u mol/L | 123.2 (70.22-609.4) | 85.2 (68.63-130.15) | 0.000 |
CCr ml/min | 38.61 (7.93-89.41) | 64.9 (23.09-106.7) | 0.000 |
UCr mmol/L | 8.03 (1.77-23.79) | 10.28 (5.2-23.57) | 0.020 |
UPCI mg/mmol | 21.91 (5.43-418.47) | 11.72 (5.22-26.24) | 0.000 |
UACI mg/mmol | 1.73 (0.18-61.72) | 0.97 (0.2-12.17) | 0.043 |
HGB g/L | 14.1 (8.7-141.6) | 124 (12.71-157.9) | 0.000 |
Glucose mmol/L | 4.86 (3.8-6.6) | 5.1 (4.31-6.69) | 0.022 |
Beta2 mikroglobulin u g/L | 99.81 (5.85-4794) | - | - |
U Protein mg/L | 225 (36.5-1517.5) | 110 (50-499) | 0.005 |
U Albumin mg/l | 17.28 (1.44-415.24) | 8.37 (2.14-232.24) | 0.197 |
Values are expressed as mean value (or percent) and median value (5th - 95th percentil)
We show the baseline characteristics of BEN study cases and control subjects in
The concentration of TBARs in plasma and urine is shown on Figure 1.The increased level of TBARS in plasma of patients with BEN was statistically significant when compared to control (
The level of AOPP in plasma and urine is shown on Figure 2.The increased level of plasma AOPP concentration in patients with BEN was statistically significant compared to the control (
The activity of XO in plasma and urine is shown on Figure 4. The decrease in plasma XO activity of BEN patients was statistically significant when compared to control (p < 0.005). The urinary level of XO activity was significantly lower in patients with BEN when compared to control (p < 0.001).
The XO values in patients with BEN and controls - serum / urine are shown on Figure 5. There was statistically lower plasma/urine XO ratio in BEN patients when compared to control (
BEN represents a disease that affects a great number of inhabitants of endemic areas. Epidemiologic findings have revealed within the last decades strongly proved that BEN is an environmentally induced disease. Aristolochia, ochratoxin, Selenium deficiency, heavy metals, are among the most important factors contributing to the etiology of BEN. However, the influence of these factor in association with urothelial cancer remains absolutely misconstrued.
The first document about the influence of food containing AA in BEN and associated cancer of upper urothelial tract was initially projected in 1969 by Ivic (34) what is now in line with the unique epidemiological features of BEN. Also inhabitants in regions suffering from BEN are exposed to relatively high concentrations of ochratoxin (OTA) (35). OTA is a potent toxin and renal carcinogen in rodents.
Heavy metals are also one of the epidemiological factors thought to be one of the main reasons for BEN development. Among them, a significant concentration in water and soil was documented for silica, lead, uranium, copper, cobalt, zinc, manganese, arsenic, titanium, barium, aluminium, chromium, strontium, cadmium, bismuth, molybdenum, nickel, tungsten, and antimony in BEN areas (36). It is well known that metal ions can produce free radicals via Fenton reaction. We have documented the influence of a heavy metal intake and ROS development in our previous study (37). The concentrations and the extent of selenium deficiency are also documented in rocks, soil, water, foodstuffs, and blood serum samples collected from endemic and non-endemic regions of BEN in the geographic area (22-24). Selenium is very important for anti-oxidative defence system.
The deoxyribonucleic acid DNA-AA adducts induced by AA produces a particular molecular signature in kidneys with aristolochic acid nephropathy. However, the pathophysiological mechanisms whereby AA results in renal injury is still unclear. Decll` eve’s et al. (38) showed that nitric oxide (NO) plays an important role in mediating aristolochic acid-induced kidney injury, leading to enhanced reactive oxygen species (ROS) and tubular cells programmed cell death. Aristolochic acid-induced oxidative stress could mediate the peritubular capillary loss and additional vascular transformation, the vital pathophysiological processes within the development of chronic nephropathy from AKI in different injury models.
Among mycotoxins, the ochratoxin A was documented to be an oxidative stress inducer. Taking into consideration all the information, the EU Food Safety Authority (EFSA) scientific panel on contaminants within the food chain gave the conclusion that there was no proof for the existence of specific OTA- DNA adducts and that the genotoxic effects of OTA were presumably due to oxidative stress injury (European Food Safety Authority 2006).
The kidneys are the extremely vulnerable organs to ROS damage, because of a long-chain-polyunsaturated fatty acids present in cell membranes. Results of our study have shown a higher level of TBARS and AOPP in patients with BEN compared to control in plasma. One of the first events in oxidative cellular injury is the oxidization of membrane lipids. Lipid hydroperoxides are non-radical intermediates derived from unsaturated fatty acids, phospholipids, glycolipids, cholesterol esters and cholesterol itself. Their formation occurs in an enzymatic or non-enzymatic reactions involving ROS. Measuring the breakdown product like TBARS, a nephrotoxic molecule which is a biological marker of oxidative stress, is the most frequently performed method for determination of lipid per-oxidation (39). In our study high level of lipid per-oxidation was statistically significant in plasma, but not in urine, what may show that systemic oxidative stress may arise because of kidney dysfunction and different uraemic toxins accumulation.
Presumably the foremost vital result in oxidative stress systemic effect and kidney injury would be the level of AOPP. Our study showed that the AOPP level is elevated in the plasma and urine of patients with BEN. Hong Yan Li and authors (40) showed that in the remnant kidney model, a higher AOPP level resulted in accelerated progression of renal injury, as proved by a marked increase of tubular fibrosis and glomerulosclerosis, additionally higher level of albuminuria, and deteriorated renal dysfunction. The finding that experimental AOPP administration raised the urinary protein excretion in sham-operated rats confirmed their direct toxic effect (41). Our results also suggest that AOPP is involved in the process of renal recast, moreover because of the subsequent fibrogenic process in the kidney. But the increase in the plasma /urine ratio in BEN patients may show on their systemic origin as well. Hong Yan Li also reported that chronic administration of AOPP during this remnant kidney model considerably raised the renal levels of AOPP, followed by magnified levels of TBARS and reduced GSHPx activity (40). Moreover, the AOPP administration evoked an imbalance of oxidation-reduction reaction in rats with intact kidney and normal renal function, suggesting that the more severe oxidative stress couldn’t attribute to the progression of excretory organ dysfunction alone. Taken together, these results recommend that AOPP additionally could be one among the inducers of oxidative stress in BEN. The close relationship between AOPP and TBARS levels or GSHPx activity and the in vitro studies showing the respiratory burst of human neutrophils that were exposed to AOPP (41) give additional proof to support the notions. Since chronic AOPP administration raised expression of TGF-1, a well-documented fibrogenic growth factor, it was a direct relation between the AOPP and the pathogenesis of nephritic inflammation and fibrosis, that’s one among the key mechanisms in kidney deterioration in patients with BEN (42,43).
ROS also can directly damage the deoxyribonucleic acid of proximal tubules and augment programmed cell death. To support this hypothesis, it had been reported in a previous study that AA depleted the antioxidant glutathione in human renal tubular cells (HK-2) leading to tubular cell death (44). Tubular cell death may be related to the oxidative stress-induced vascular effects of AA. Intra-renal oxidative stress exacerbates smooth muscle cell proliferation of the afferent arterioles and promotes renin-angiotensin system activation. It was documented that ischaemic injury as a part of AA-induced AKI related to reduced NO levels, and these oxidative stress-induced vascular changes may also lead to tubular cell death (45). Aristolochic acid can directly reduce the concentration of NO and can mediate the peritubular capillary loss additionally as vascular transforming. Exactly how tubulointerstitial fibrosis develops in AAN is unclear, however, supposed mechanisms include raised production of profibrotic growth factors (e.g. transforming growth factor-β), cell cycle arrest in G2 and accumulated inflammation. Oxidative stress might play a role in one or additional of those mechanisms, resulting in progressive loss of renal function.
Another important question is what is the most probable source of free radical production. AA is directly inducer of oxidative stress, but that’s certainly not the main cause of it. In the literature there’s a great deal of evidence regarding the role of enhanced ROS within the kidney and protective role of antioxidants and ROS scavengers in IR injury-induced. (46-48). Indeed, fibrotic kidney is under ischaemia. However, this mechanism occurs only in the last stages of the disease. Another potential source of ROS could be uraemic toxins in patients with CKD. A higher level of those toxins promotes systemic inflammation via priming polymorphonuclear leukocytes and stimulating CD-8+ cells (49). An important issue of ROS production is that of the bio-incompatibility of HD systems. In general, two major parts of a HD system will contribute to oxidative stress: the dialyzer membrane and trace endotoxins within the dialysate (50,51). However, it’s unlikely the explanation in this disease because not all patients with BEN were uremic ones. Additionally, our hypothesis was that the source of free radicals could be xanthine oxidase activity, since liver, gut and kidney represent an XO-rich organs. XO-induced ROS production has been documented for development of ischaemic intestinal, hepatic and nephritic injury (52). Additionally, the long-term treatment with XO inhibitors achieved reno-protective advantages in individuals with a non-advanced chronic renal disorder (53). A large body of clinical proofs nowadays pointed uric acid as a possible therapeutic target for slowing down CKD progression (54). Gouty patients and even people with symptomless hyperuricemia have a sustained risk of developing renal damage; equally, in patients with obvious CKD, steady elevated uric acid levels might contribute to worsening renal function (55). Since ROS are strictly related to kidney pathology (56), it had been reported that XOR redox, which is outlined as the quantitative relation of XO to total XOR (XO and XDH), changes the oxidative state are in correlation with kidney pathology (57).
Xanthine oxidoreductase (XOR) is a key enzyme of urate synthesis, which catalyses the reaction of hypo-xanthine to xanthine and catalyses the oxidization of xanthine to the final product uric acid. We find XOR not only as xanthine dehydrogenase (XDH) type but also as XO form, which uses an oxygen molecule as an electron acceptor and generates ROS like superoxide anion radicals and hydrogen peroxide. We have already proven that XO activity is one of the possible reasons for ROS liberation in in experimental kidney damage (37). It’s also documented that XO is responsible for ischaemia injury and fibrosis (58).
All the above-mentioned findings led to the expectancy of a higher activity of XO in BEN patients since there are a lot of causes that would raise its activity, but it was not a case. Our results showed lower XO activity in patients with BEN compared to healthy control subjects (Figure 3). Plasma/urine ratio showed even more significant differences, what may point a systemic origin of the enzyme. Indeed, there are only a few patients with BEN who suffered from gout as a chronic disease together with BEN. One of possible reasons would be the leak of actual substrates for XO, like urine nucleotides. Lower kidney mass, accompanied with diminished functional tissue, may be responsible for lower substrate level. Further studies showed the importance of Aristolochic acid in formation of DNA-AA adducts (59). The resulting adenine (AL-N6-dA) and guanine (ALN2- dg,) adducts mis incorporate adenine throughout replication (20,21). Specifically, the intrinsic twisted conformation of ALII-N2-dG induces destabilizing distortions to deoxyribonucleic acid at the lesion site, reduces van der Waals (stacking) interactions with the neighbouring base pairs and enhances the helical dynamics at the damaged site (60). Above-mentioned changes affect the purine nucleotide structure in patients with BEN, probably making them as conformationally not recognizable for XO, because Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides.
Based on the results of this research, it can be concluded that the level of lipid peroxide and AOPP are significantly increased BEN patients, and that systemic ROS production is involved in pathogenesis of BEN. XO activity is not the source of ROS in kidney tissue, most probably because of the diminished kidney functional tissue mass and AA-induced changes in purine nucleotide conformation.
This work was supported by the Ministry of Science and Technological development, Republic of Serbia (Projects 43012, 31060, and 41018), and by Faculty of Medicine, University in Nis, Internal scientific project number 45, and Serbian Academy of Sciences and Arts, a branch in Nis (Projects О-06-17 and О-07-17).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.