Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
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Urotensin-II level and its association with oxidative stress in early diabetic nephropathy Suzan Tabur a, Hakan Korkmaz a,⁎, Mehmet Ali Eren b, Elif Oğuz c, Tevfik Sabuncu b, Nurten Aksoy d a
Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, Gaziantep University, Sahinbey, Gaziantep 27100, Turkey Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, Harran University, 63300 Sanliurfa, Turkey Department of Medical Pharmacology, Faculty of Medicine, Harran University, 63300 Sanliurfa, Turkey d Department of Clinical Biochemistry, Faculty of Medicine, Harran University, 63300 Sanliurfa, Turkey b c
a r t i c l e
i n f o
Article history: Received 24 March 2014 Received in revised form 24 July 2014 Accepted 24 July 2014 Available online xxxx Keywords: Diabetic nephropathy Urotensin-II Oxidative stress Paraoxonase Arylesterase
a b s t r a c t Objective: Diabetic nephropathy is the most common cause of end stage renal failure. Early treatment of diabetic nephropathy depends on understanding the underlying mechanisms of the disease. In this study we investigated the role of U-II in early nephropathy and ıts association with oxidative stress, paraoxonase (PON)-1 and arylesterase. Research design and methods: Twenty-three diabetic patients with microalbuminuria, 23 diabetic patients with normoalbuminuria and 25 healthy individuals were enrolled in the study. Serum total antioxidant status (TAS), total oxidant status (TOS), PON-1, arylesterase, and urotensin-II (U-II) levels were measured. Oxidative stress index (OSI) percent ratio of TOS to TAS level was accepted as OSI. Results: Serum U-II levels were higher in the microalbuminuric diabetes group compared to the normoalbuminuric diabetic group and the healthy control group (p = 0.009 and p = 0.0001, respectively). Normoalbuminuric diabetic group's U-II levels were significantly higher compared to those of the healthy control group (p = 0.0001). Correlation analysis yielded that plasma U-II levels are negatively correlated to TAS, arylesterase, and PON-1 levels (r = −0.395, p = 0.001; r = −0.291, p = 0.014; and r = −0.279, p = 0.018, respectively) and that they had a positive correlation with OSI levels (r = 0.312, p = 0.008). These associations were confirmed in the multiple regression analysis. The results of multiple logistic regression analysis showed that oxidative stress is important in the development of microalbuminuria. Conclusion: The data of this study reveal that increased serum U-II has a role in the development of diabetic nephropathy. This effect of U-II may be related to high levels oxidative stress parameters. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Diabetic nephropathy is one of the important complications of diabetes mellitus (DM) and is the most common cause of end stage renal failure in clinical practice (Reutens, 2013). Early diagnosis of diabetic nephropathy depends on understanding the underlying mechanisms. Though microalbuminuria has been identified as the most effective indicator of early diabetic nephropathy, some structural changes might have already occurred by the time microalbuminuria is detected (Araki et al., 2008). It is highly important to prevent development of microalbuminuria.
Conflict of Interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. ⁎ Corresponding author at: Gaziantep University, Faculty of Medicine, Department of Internal Medicine, Division of Endocrinology, 27100 Sahinbey, Gaziantep, Turkey. Tel.: +90 342 341 66 89; fax: +90 342 360 60 60. E-mail address: [email protected] (H. Korkmaz).
Urotensin-II (U-II) is recognized as the strongest vasoconstrictor detected among mammals to this day (Ross, McKendy, & Giaid, 2010). U-II and its receptors are found in various tissues such as heart, brain, kidney, smooth muscle, and endothelium (Barrette & Schwertani, 2012; Onan, Hannan, & Thomas, 2004). It is a significant mediator in renal diseases (Adebiyi, 2014; Balat, Karakök, Yilmaz, & Kibar, 2007). Various studies have shown that diabetic patients have increased serum U-II levels (Ong, Wong, & Cheung, 2008; Totsune et al., 2003). Similarly, in diabetic nephropathy patients, U-II and its receptors' expression has been detected to have increased (Langham et al., 2004). However, there is not sufficient information on U-II's exact role in diabetic nephropathy development. Oxidative stress increase is an important condition in DM; and it is known to be an important cause of diabetic nephropathy development (King & Loeken, 2004). It is suspected that there is an imbalance between the oxidant and anti-oxidant mediators prior to development of renal lesions, and that the oxidation level increases as the disease progresses (Piarulli et al., 2009). Paraoxonase-1 (PON-1), which has paraoxonase (PON) and arylesterase activities, is a high-
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Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
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S. Tabur et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
density lipoprotein cholesterol (HDL-C) bound antioxidant enzyme that protects low-density lipoprotein cholesterol (LDL-C) against oxidative damage. Low levels of PON and arylesterase, antioxidant enzymes, have been reported in situations in which reactive oxygen radicals increase and oxidative stress occurs (Wegner, Piorunska-Stolzmann, Araszkiewicz, Zozulińska-Ziółkiewicz, & Wierusz-Wysocka, 2011). In this study, we investigated the role of serum U-II in early nephropathy and its association with oxidative stress. 2. Materials and methods The study was initiated upon obtaining approval from ethics Committee of Harran University Faculty of Medicine numbered (15.06.2009/07-18). All participants were informed and an informed consent was obtained from all of them prior to the study. 2.1. Patient group and study protocol A total of 71 participants were included in the study: 23 diabetic patients with microalbuminuria (median age = 50; IQR: 9; 12 females, 11 males); 23 diabetic patients with normoalbuminuria (median age = 46; IQR: 9; 13 females, 10 males); and 25 healthy individuals (median age = 47; IQR: 11.5; 15 females, 10 males). People with systemic diseases such as infectious diseases, inflammatory diseases, hypertension, liver failure, cardiovascular diseases, malignancies, neurodegenerative diseases, cerebrovascular diseases; those on antioxidants such as antihypertensive medications, lipid-lowering medications, and vitamin E; and smokers were excluded from the study. Seven patients with microalbuminuria and 2 patients with normoalbuminuria were taking insulin therapy. Other patients were taking at least 2 oral antidiabetic drugs. 2.2. Evaluation of nephropathy and duration of diabetes mellitus Mean urine albumin/creatinine index was measured in the spot urine, collected on 3 different days. Urine contaminated with bacteria, red blood cells, and white blood cells were removed. Urinary albumin concentration was measured via latex turbidimetric immunoassay method using commercial kits. When albumin/creatinine = 30–300 mg/g in type 2 DM patients, it was considered as microalbuminuria. The staging criteria recommended by Mogensen et al. were used for early diabetic renal disease diagnosis (Mogensen, Christensen, & Vittinghus, 1983). For the duration of DM, the time of initial symptoms associated with the disease was considered beginning of the disease. If there were no symptoms, the time of diagnosis was considered as the beginning. The American Diabetes Association’s, 2010 criteria were used for DM diagnosis (ADA, 2010). 2.3. Measurements Systolic blood pressure (SBP), diastolic blood pressure (DBP), height, and weight of each participant were measured. Body mass index (BMI) was calculated as body mass (kg)/height (m) 2. Blood samples were collected in the morning hours after an 8-hour fasting period. Serum samples were stored at − 80 °C until total antioxidant status (TAS), total oxidant status (TOS), PON, arylesterase, and U-II levels were measured. Urine microalbumin and creatinine values were measured via turbidimetric method by a Cobas Integra 800 model auto-analyzer (Roche®). Hemoglobin A1c (HbA1c) levels were tested by using the Celldyn 3700 (Abbott, ®USA) auto-analyzer commercial kit. Serum urea and creatinine values were measured spectrophotometrically by routine biochemical methods using Cobas Integra 800 model auto-analyzer (Roche®). Serum triglyceride (TG), total cholesterol, LDL-C, and HDL-C concentrations were measured using an auto-analyzer (Aeroset, Abbott, USA) commercial kit (Abbott, USA).
2.4. Measurement of total oxidant/antioxidant status Serum TOS was determined using a novel automated measurement method developed by Erel (2005). Oxidants present in the study sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed as mmol H2O2 Equiv./l. Serum TAS was determined using a novel automated measurement method developed by Erel (2004). In the method, hydroxyl radical, the most potent biological radical, is produced first. In the assay, reagent 1 containing ferrous ion solution is mixed with reagent 2, which contains hydrogen peroxide. The sequentially produced radicals, such as brown colored dianisidinyl radical cation produced by the hydroxyl radical, are also potent radicals. The anti-oxidative effect of the study sample against the potent-free radical reactions, which are initiated by the produced hydroxyl radical, is measured. The assay has excellent precision values, lower than 3%, and the results are expressed as mmol Trolox Equiv./l. Oxidative stress index percent ratio of TOS to TAS level was accepted as OSI [OSI (arbitrary unit) = TOS (mmol H2O2 Equiv./l)/TAS (mmol Trolox Equiv./l)] (Bolukbas et al., 2005). 2.5. Measurements of PON and arylesterase activities PON and arylesterase activities were measured with commercially available kits (Relassay, Gaziantep, Turkey). PON measurement was performed either in the presence (salt-stimulated) or in the absence of NaCl. Paraoxon hydrolysis rate (diethyl-p-nitrophenyl phosphate) was measured by monitoring increased absorption at 412 nm at 37 °C. The amount of generated p-nitrophenol was calculated from the molar absorption coefficient at pH 8.5, which was 18.290/M per cm (Eckerson, Wyte, & La Du, 1983). PON activity was expressed as U/l serum. The coefficient of variation (CV) for individual samples was 1.8%. Arylesterase activity was measured using phenyl acetate as substrate. Enzymatic activity was calculated from the molar absorption coefficient of the produced phenol, 1310/M per cm. One unit of arylesterase activity was defined as 1 mmol phenol generated per minute under the above conditions and expressed as U/l (Haagen & Brock, 1992). The CV for individual serum samples was 4.1%. The sensitivities of both tests were over 98%. 2.6. Measurement of U-II level Serum U-II levels were determined by Sandwich ELISA (Phoenix Pharmaceuticals®, USA). 2.7. Statistical analysis Shapiro–Wilk test was used to test continuous variables for normality. Measurements of normally distributed variables (TAS, OSI, HDL, LDL, urea and arylesterase) are presented as mean ± standard deviation. Those with non-normal distributions are presented as median and interquartile range (IQR). Student's t-test was used in comparison of 2 independent groups of normally distributed variables; one-way analysis of variance (ANOVA) test was used when comparing more than 2 groups; and LSD test was used for paired comparisons to identify which group the difference was caused by. For non-normally distributed variables, Mann–Whitney U test was used to compare 2 independent groups and Kruskall Wallis test was used to compare more than 2 independent groups. DUNN test was used for post-hoc comparisons. Spearman correlation analysis was done to identify
Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
S. Tabur et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
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Table 1 Clinical and metabolic parameters of the microalbuminuric diabetic, normoalbuminuric diabetic and healthy control groups. Parameter
Microalbuminuric diabetic (n = 23)
Normoalbuminuric diabetic (n = 23)
Healthy control (n = 25)
p value
Age (years) Sex (F/M) BMI (kg/m2) Duration of DM (years) HbA1c (%) Insulin use SBP (mmHg) DBP (mmHg) FPG (mg/dl) LDL (mg/dl) HDL (mg/dl) BUN (mg/dl) Cr (mg/dl) Urinary microalbumin/Cr (mg/L) Serum U-II (ng/ml) TAS (μmol H2O2) TOS (mmol Equiv./l) OSI (arbitrary unit) PON-1 (U/ml) Arylesterase (U/ml)
50 ± 9a 12/11 27.43 ± 3.23a 4 ± 7a 8.50 ± 3.50a 7 120 ± 20a 80 ± 20a 168 ± 94a 110.17 ± 26.21 34.73 ± 6.47 31.17 ± 8.25 0.82 ± 0.30a 52 ± 68* 60 ± 26a,b,c 0.90 ± 0.16 19.05 ± 8.3a 2.27 ± 0.46 105 ± 122a 196.44 ± 27.87
46 ± 9a 13/10 27.28 ± 3.13a 4 ± 5a 8.80 ± 4.10a 2 120 ± 20a 80 ± 20a 170 ± 104a 107.95 ± 28.37 36.34 ± 6.04 26.65 ± 7.05 0.83 ± 0.20a 11 ± 9a 46 ± 22a,d 0.99 ± 0.11 15.03 ± 2.68a 1.65 ± 0.46 113 ± 101a 202.04 ± 38.94
47 ± 11.50a 15/10 26.70 ± 3.02a
0.270 0.863 0.292 0.095 0.767 0.066 0.677 0.648 0.0001 0.375 0.064 0.070 0.622 0.0001 0.0001 0.0001 0.0001 0.0001 0.005 0.016
120 ± 20a 75 ± 20a 87 ± 20a 119.08 ± 31.99 39.40 ± 7.85 27.16 ± 6.12 0.90 ± 0.10a 8 ± 11a 13 ± 6a 1.11 ± 0.12 15.44 ± 2.14a 1.38 ± 0.22 194 ± 139.5 223.99 ± 34.80
DM, diabetes mellitus; BMI, body mass index; FPG, fasting plasma glucose; HDL/LDL, high-density lipoprotein/low-density lipoprotein; BUN, blood urea nitrogen; Cr, creatinin; OSI, oksidative stress index; PON, paraoxonase; SBP/DBP, systolic blood pressure/diastolic blood pressure; TAS, total antioksidant status; TOS, total oxidative status. a Data in which non-parametric tests were used and expressed as median (Inter quarter range) p b 0.0001. b Microalbuminuric diabetic versus normalbuminurik diabetic; p b 0.0001. c Microalbuminuric diabetic versus control; p b 0.0001. d Normalbuminuric diabetic versus control; p b 0.0001.
associations between the parameters. Multiple linear regression analysis was conducted to identify the variables impacting the U-II level. Multiple logistic regression analysis was done to evaluate the relationship between microalbuminuria and TAS, TOS, PON, arylesterase, and U-II in diabetic patients. SPSS for Windows version 15 software was used for statistical analyses. The level of significance was set at p ≤ 0.05.
3. Results The mean age of all 3 groups and their gender distribution were similar (p = 0.270 and p = 0.863, respectively). BMI, SBP, DBP, LDL-C, HDL-C, blood urea nitrogen (BUN), and creatinine levels were not significantly different across the groups (p = 0.292, p = 0.677, p = 0.648, p = 0.375, p = 0.064, p = 0.070, and p = 0.622, respectively). The diabetic patient groups with and without microalbuminuria were not different in terms of duration of DM, insulin use, and HbA1c levels (p = 0.095, p = 0.066, and p = 0.767, respectively; Table 1). TAS level was lower in the microalbuminuric and normoalbuminuric diabetic groups compared to the healthy control group (p = 0.0001 and p = 0.006, respectively). While the TAS level was lower in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group, the difference was not significant (p = 0.093). OSI levels were higher in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group and the healthy control group (p = 0.0001 and p = 0.0001, respectively). The OSI levels in the normoalbuminuric group were higher than those in the healthy control group and the difference could not reach the level of significance (p = 0.054). TOS levels were higher in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group and the healthy control group (p = 0.002 and p = 0.0001, respectively). However, there was no significant difference between the normoalbuminuric group and the healthy control group in terms of TOS levels (p = 0.861). PON levels were lower in the microalbuminuric diabetic group and the normoalbuminuric diabetic group compared to the healthy control group (p = 0.005 and p = 0.005, respectively). The groups with and without microalbuminuria did not differ significantly with respect to PON levels (p = 0.930).
Serum U-II levels were higher in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group and the healthy control group (p = 0.009 and p = 0.0001, respectively). Normoalbuminuric diabetic group's serum U-II levels were significantly higher compared to the healthy control group (p = 0.0001; Fig. 1). Arylesterase levels were lower in the microalbuminuric diabetic group compared to the healthy control group (p = 0.021). On the other hand, there was no significant difference between diabetic groups with and without microalbuminuria or between the normoalbuminuric group and the healthy control group (p = 0.844 and p = 0.089, respectively). Correlation analysis yielded that serum U-II levels are weak negatively correlated to TAS, arylesterase, and PON levels (r = −0.395, p = 0.001; r = −0.291, p = 0.014; and r = −0.279, p = 0.018, respectively) and that they had a weak positive correlation with TOS levels (r = 0.312, p = 0.008; Figs. 2–5). These associations were confirmed in the multiple regression analysis (Table 2). Multiple logistic regression analysis results demonstrated the important association between microalbuminuria development and
Fig. 1. Serum concentrations of U-II in healthy volunteers and diabetic patients with or without microalbuminuria.
Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
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S. Tabur et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
Spearman correlation analysis (r=-0.395 , p= 0.001 )Total antioxidant status, TAS
Spearman correlation analysis (r=-0.279 , p= 0.018 ), Paraoxonase, PON
Fig. 2. Correlation between serum U-II levels and TAS. Spearman correlation analysis (r = −0.395, p = 0.001). Total antioxidant status, TAS.
Fig. 4. Correlation between serum U-II levels and PON. Spearman correlation analysis (r = −0.279, p = 0.018). Paraoxonase, PON.
oxidative stress and serum U-II levels in diabetic individuals (3).
the dilated tubular epithelium or damaged tubules. It has shown that the U-II has a direct role in tubular toxicity (Langham et al., 2004). A 24.3% decrease has been established in daily albumin excretion of diabetic nephropathy patients with macroalbuminuria by administration of urotensin receptor antagonist, palosuran (Sidharta et al., 2006). Similarly, in diabetic rats, palosuran has been demonstrated to prevent the progressive increase in albuminuria, renal dysfunction, and increases in tubular and tubulointerstitial lesions (Clozel, Hess, Qiu, Ding, & Rey, 2006). There is no study evaluating the effect of urotensin receptor antagonists in early diabetic nephropathy patients. Different mechanisms are activated as diabetic nephropathy progresses. We believe that treatment effectiveness could be higher in early stage. Various studies have shown that the oxidative stress develops due to oxidant and antioxidant balance impairments (Bedard & Krause, 2007). The role of oxidative stress in development of diabetic nephropathy has been demonstrated (King & Loeken, 2004). TAS, TOS, and OSI levels were analyzed as oxidative stress markers in this study. TAS level was lower in the microalbuminuric and normoalbuminuric diabetic groups compared to the healthy control group (p = 0.0001 and p = 0.005, respectively). TOS and OSI levels were higher in the microalbuminuric diabetic group compared to
4. Discussion This is the first study demonstrating the association between the increased serum U-II levels and early nephropathy in type 2 DM patients. In addition to serum U-II levels, increased oxidative stress is also an independent risk factor for development of early diabetic nephropathy. Similar to our results, Totsune et al. have also detected higher serum U-II levels in diabetics compared to healthy control patients. However, they did not detect a difference between the serum U-II levels of diabetic patients with and without proteinuria (Totsune et al., 2003). The difference between our results and theirs may be due to the low sample size (16 diabetic patients) of their study and that they had taken 100 mg albumin/g Cr as the cut-off point for proteinuria. In this case, patients with microalbuminuria are also placed in the group without proteinuria. In this study, we took 30–300 mg albumin/g Cr value as indication of microalbuminuria because microalbuminuria is the best indicator of early nephropathy. It has been shown that the expression of urotensin receptors in kidneys of diabetic patients is increased. This is observed especially in
Spearman correlation analysis (r= -0.291 , p= 0.014 )
Fig. 3. Correlation between serum U-II levels and arylesterase. Spearman correlation analysis (r = −0.291, p = 0.014).
Spearman correlation analysis (r=0.312 , p=0.008 ), TOS, total oxidant status
Fig. 5. Correlation between serum U-II levels and TOS. Spearman correlation analysis (r = 0.312, p = 0.008). TOS, total oxidant status.
Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
S. Tabur et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx Table 2 Multiple linear regression analysis between U-II and oxidant and antioxidant markers.
TAS TOS Arylesterase PON-1
β coefficients
p value
−0.879 1.296 −0.080 −0.027
0.016⁎ 0.016⁎ 0.451 0.035⁎
TAS, total antioxidant status; TOS, total oxidant status; PON, paraoxonase. ⁎ Significant at p b 0.05.
the normoalbuminuric diabetic group and the healthy control group (p = 0.002; p = 0.0001; p = 0.0001; p = 0.0001, respectively). Though the TOS and OSI levels were higher in the normoalbuminuric diabetic group compared to the healthy control group, the differences were not significant (p = 0.861 and p = 0.054, respectively). This shows that in the impairment in the balance of oxidants and antioxidants, the decrease in the antioxidants occurs prior to the increase in the oxidants. Continued oxidant level increase is thought to contribute to the progression into nephropathy. In our study, we evaluated the correlation between oxidative stress and serum U-II levels as well. There was a negative correlation between serum UII level and TAS and a positive correlation between TOS and OSI (p = 0.016 and p = 0.016, respectively). Based on the multiple regression analysis results, oxidative stress was shown to be an independent risk factor responsible for increases in the serum U-II levels (Table 3). PON-1 is an important molecule in atherosclerosis pathogenesis. Studies have determined lower PON1 activities in type 1 and 2 DM compared to healthy individuals (Ferretti, Bacchetti, Busni, Rabini, & Curatola, 2004; Flekac, Skrha, Zidková, Lacinová, & Hilgertová, 2008; Karabina, Lehner, Frank, Parthasarathy, & Santanam, 2005). In accordance with other studies, the PON1 level was lower in both of the diabetic groups with and without microalbuminuria compared to the healthy control group (p = 0.005 and p = 0.005, respectively). The role of PON1 activities in the development of DM-related complications has been investigated. A negative correlation was detected between the PON1 activities and presence of vascular complications (Karabina et al., 2005). Diabetic patients with neuropathy were demonstrated to have lower PON1 levels compared to diabetes patients without neuropathy (Poh & Muniandy, 2010). Another study has shown an association between PON1 expression and development of diabetic nephropathy in type 2 DM patients (Sun, 2010). In this study, the PON and arylesterase levels were lower in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group, but the difference was not significant (p = 0.930 and p = 0.844). Correlation analysis yielded that the U-II levels were negatively correlated to arylesterase and PON (r = − 0.291, p = 0.014; r = − 0.279, p = 0.018, respectively). Multiple regression analysis has shown that decreasing levels of PON is an independent risk factor responsible for increases in the serum U-II levels (p = 0.035). Consequently, U-II system has a role in the development of early diabetic nephropathy in type 2 DM. U-II system is affected by oxidative stress. Further studies on this topic will help to better
Table 3 Results of logistic regression models predicting microalbuminuria.
TAS TOS Arylesterase PON-1
OR (95% CI)
p value
0.001 1.419 1.005 0.991
0.011⁎ 0.007⁎ 0.720 0.161
(0.0001–0.134) (1.098–1.834) (0.980–1.030) (0.979–1003)
TOS, total oxidant status; PON, paraoxonase. ⁎ Significant at p b 0.05, TAS, total antioxidant status.
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Pharmacodynamics and pharmacokinetics of the urotensin II receptor antagonist palosuran in macroalbuminuric, diabetic patients. Clinical pharmacology and therapeutics, 80, 245–256. Sun, Y. M. (2010). Study of SOD and PON-1 expression in type 2 diabetes mellitus nephropathy and its clinical significance. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 26, 1120–1121. Totsune, K., Takahashi, K., Arihara, Z., Sone, M., Ito, S., & Murakami, O. (2003). Increased plasma urotensin II levels in patients with diabetes mellitus. Clinical Science (London, England : 1979), 104, 1–5. Wegner, M., Piorunska-Stolzmann, M., Araszkiewicz, A., Zozulińska-Ziółkiewicz, D., & Wierusz-Wysocka, B. (2011). Evaluation of paraoxonase 1 arylesterase activity and lipid peroxide levels in patients with type 1 diabetes. Polskie Archiwum Medycyny Wewnętrznej, 121, 448–454.
Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
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Urotensin-II level and its association with oxidative stress in early diabetic nephropathy Suzan Tabur a, Hakan Korkmaz a,⁎, Mehmet Ali Eren b, Elif Oğuz c, Tevfik Sabuncu b, Nurten Aksoy d a
Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, Gaziantep University, Sahinbey, Gaziantep 27100, Turkey Division of Endocrinology, Department of Internal Medicine, Faculty of Medicine, Harran University, 63300 Sanliurfa, Turkey Department of Medical Pharmacology, Faculty of Medicine, Harran University, 63300 Sanliurfa, Turkey d Department of Clinical Biochemistry, Faculty of Medicine, Harran University, 63300 Sanliurfa, Turkey b c
a r t i c l e
i n f o
Article history: Received 24 March 2014 Received in revised form 24 July 2014 Accepted 24 July 2014 Available online xxxx Keywords: Diabetic nephropathy Urotensin-II Oxidative stress Paraoxonase Arylesterase
a b s t r a c t Objective: Diabetic nephropathy is the most common cause of end stage renal failure. Early treatment of diabetic nephropathy depends on understanding the underlying mechanisms of the disease. In this study we investigated the role of U-II in early nephropathy and ıts association with oxidative stress, paraoxonase (PON)-1 and arylesterase. Research design and methods: Twenty-three diabetic patients with microalbuminuria, 23 diabetic patients with normoalbuminuria and 25 healthy individuals were enrolled in the study. Serum total antioxidant status (TAS), total oxidant status (TOS), PON-1, arylesterase, and urotensin-II (U-II) levels were measured. Oxidative stress index (OSI) percent ratio of TOS to TAS level was accepted as OSI. Results: Serum U-II levels were higher in the microalbuminuric diabetes group compared to the normoalbuminuric diabetic group and the healthy control group (p = 0.009 and p = 0.0001, respectively). Normoalbuminuric diabetic group's U-II levels were significantly higher compared to those of the healthy control group (p = 0.0001). Correlation analysis yielded that plasma U-II levels are negatively correlated to TAS, arylesterase, and PON-1 levels (r = −0.395, p = 0.001; r = −0.291, p = 0.014; and r = −0.279, p = 0.018, respectively) and that they had a positive correlation with OSI levels (r = 0.312, p = 0.008). These associations were confirmed in the multiple regression analysis. The results of multiple logistic regression analysis showed that oxidative stress is important in the development of microalbuminuria. Conclusion: The data of this study reveal that increased serum U-II has a role in the development of diabetic nephropathy. This effect of U-II may be related to high levels oxidative stress parameters. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Diabetic nephropathy is one of the important complications of diabetes mellitus (DM) and is the most common cause of end stage renal failure in clinical practice (Reutens, 2013). Early diagnosis of diabetic nephropathy depends on understanding the underlying mechanisms. Though microalbuminuria has been identified as the most effective indicator of early diabetic nephropathy, some structural changes might have already occurred by the time microalbuminuria is detected (Araki et al., 2008). It is highly important to prevent development of microalbuminuria.
Conflict of Interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. ⁎ Corresponding author at: Gaziantep University, Faculty of Medicine, Department of Internal Medicine, Division of Endocrinology, 27100 Sahinbey, Gaziantep, Turkey. Tel.: +90 342 341 66 89; fax: +90 342 360 60 60. E-mail address: [email protected] (H. Korkmaz).
Urotensin-II (U-II) is recognized as the strongest vasoconstrictor detected among mammals to this day (Ross, McKendy, & Giaid, 2010). U-II and its receptors are found in various tissues such as heart, brain, kidney, smooth muscle, and endothelium (Barrette & Schwertani, 2012; Onan, Hannan, & Thomas, 2004). It is a significant mediator in renal diseases (Adebiyi, 2014; Balat, Karakök, Yilmaz, & Kibar, 2007). Various studies have shown that diabetic patients have increased serum U-II levels (Ong, Wong, & Cheung, 2008; Totsune et al., 2003). Similarly, in diabetic nephropathy patients, U-II and its receptors' expression has been detected to have increased (Langham et al., 2004). However, there is not sufficient information on U-II's exact role in diabetic nephropathy development. Oxidative stress increase is an important condition in DM; and it is known to be an important cause of diabetic nephropathy development (King & Loeken, 2004). It is suspected that there is an imbalance between the oxidant and anti-oxidant mediators prior to development of renal lesions, and that the oxidation level increases as the disease progresses (Piarulli et al., 2009). Paraoxonase-1 (PON-1), which has paraoxonase (PON) and arylesterase activities, is a high-
http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011 1056-8727/$© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
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S. Tabur et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
density lipoprotein cholesterol (HDL-C) bound antioxidant enzyme that protects low-density lipoprotein cholesterol (LDL-C) against oxidative damage. Low levels of PON and arylesterase, antioxidant enzymes, have been reported in situations in which reactive oxygen radicals increase and oxidative stress occurs (Wegner, Piorunska-Stolzmann, Araszkiewicz, Zozulińska-Ziółkiewicz, & Wierusz-Wysocka, 2011). In this study, we investigated the role of serum U-II in early nephropathy and its association with oxidative stress. 2. Materials and methods The study was initiated upon obtaining approval from ethics Committee of Harran University Faculty of Medicine numbered (15.06.2009/07-18). All participants were informed and an informed consent was obtained from all of them prior to the study. 2.1. Patient group and study protocol A total of 71 participants were included in the study: 23 diabetic patients with microalbuminuria (median age = 50; IQR: 9; 12 females, 11 males); 23 diabetic patients with normoalbuminuria (median age = 46; IQR: 9; 13 females, 10 males); and 25 healthy individuals (median age = 47; IQR: 11.5; 15 females, 10 males). People with systemic diseases such as infectious diseases, inflammatory diseases, hypertension, liver failure, cardiovascular diseases, malignancies, neurodegenerative diseases, cerebrovascular diseases; those on antioxidants such as antihypertensive medications, lipid-lowering medications, and vitamin E; and smokers were excluded from the study. Seven patients with microalbuminuria and 2 patients with normoalbuminuria were taking insulin therapy. Other patients were taking at least 2 oral antidiabetic drugs. 2.2. Evaluation of nephropathy and duration of diabetes mellitus Mean urine albumin/creatinine index was measured in the spot urine, collected on 3 different days. Urine contaminated with bacteria, red blood cells, and white blood cells were removed. Urinary albumin concentration was measured via latex turbidimetric immunoassay method using commercial kits. When albumin/creatinine = 30–300 mg/g in type 2 DM patients, it was considered as microalbuminuria. The staging criteria recommended by Mogensen et al. were used for early diabetic renal disease diagnosis (Mogensen, Christensen, & Vittinghus, 1983). For the duration of DM, the time of initial symptoms associated with the disease was considered beginning of the disease. If there were no symptoms, the time of diagnosis was considered as the beginning. The American Diabetes Association’s, 2010 criteria were used for DM diagnosis (ADA, 2010). 2.3. Measurements Systolic blood pressure (SBP), diastolic blood pressure (DBP), height, and weight of each participant were measured. Body mass index (BMI) was calculated as body mass (kg)/height (m) 2. Blood samples were collected in the morning hours after an 8-hour fasting period. Serum samples were stored at − 80 °C until total antioxidant status (TAS), total oxidant status (TOS), PON, arylesterase, and U-II levels were measured. Urine microalbumin and creatinine values were measured via turbidimetric method by a Cobas Integra 800 model auto-analyzer (Roche®). Hemoglobin A1c (HbA1c) levels were tested by using the Celldyn 3700 (Abbott, ®USA) auto-analyzer commercial kit. Serum urea and creatinine values were measured spectrophotometrically by routine biochemical methods using Cobas Integra 800 model auto-analyzer (Roche®). Serum triglyceride (TG), total cholesterol, LDL-C, and HDL-C concentrations were measured using an auto-analyzer (Aeroset, Abbott, USA) commercial kit (Abbott, USA).
2.4. Measurement of total oxidant/antioxidant status Serum TOS was determined using a novel automated measurement method developed by Erel (2005). Oxidants present in the study sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed as mmol H2O2 Equiv./l. Serum TAS was determined using a novel automated measurement method developed by Erel (2004). In the method, hydroxyl radical, the most potent biological radical, is produced first. In the assay, reagent 1 containing ferrous ion solution is mixed with reagent 2, which contains hydrogen peroxide. The sequentially produced radicals, such as brown colored dianisidinyl radical cation produced by the hydroxyl radical, are also potent radicals. The anti-oxidative effect of the study sample against the potent-free radical reactions, which are initiated by the produced hydroxyl radical, is measured. The assay has excellent precision values, lower than 3%, and the results are expressed as mmol Trolox Equiv./l. Oxidative stress index percent ratio of TOS to TAS level was accepted as OSI [OSI (arbitrary unit) = TOS (mmol H2O2 Equiv./l)/TAS (mmol Trolox Equiv./l)] (Bolukbas et al., 2005). 2.5. Measurements of PON and arylesterase activities PON and arylesterase activities were measured with commercially available kits (Relassay, Gaziantep, Turkey). PON measurement was performed either in the presence (salt-stimulated) or in the absence of NaCl. Paraoxon hydrolysis rate (diethyl-p-nitrophenyl phosphate) was measured by monitoring increased absorption at 412 nm at 37 °C. The amount of generated p-nitrophenol was calculated from the molar absorption coefficient at pH 8.5, which was 18.290/M per cm (Eckerson, Wyte, & La Du, 1983). PON activity was expressed as U/l serum. The coefficient of variation (CV) for individual samples was 1.8%. Arylesterase activity was measured using phenyl acetate as substrate. Enzymatic activity was calculated from the molar absorption coefficient of the produced phenol, 1310/M per cm. One unit of arylesterase activity was defined as 1 mmol phenol generated per minute under the above conditions and expressed as U/l (Haagen & Brock, 1992). The CV for individual serum samples was 4.1%. The sensitivities of both tests were over 98%. 2.6. Measurement of U-II level Serum U-II levels were determined by Sandwich ELISA (Phoenix Pharmaceuticals®, USA). 2.7. Statistical analysis Shapiro–Wilk test was used to test continuous variables for normality. Measurements of normally distributed variables (TAS, OSI, HDL, LDL, urea and arylesterase) are presented as mean ± standard deviation. Those with non-normal distributions are presented as median and interquartile range (IQR). Student's t-test was used in comparison of 2 independent groups of normally distributed variables; one-way analysis of variance (ANOVA) test was used when comparing more than 2 groups; and LSD test was used for paired comparisons to identify which group the difference was caused by. For non-normally distributed variables, Mann–Whitney U test was used to compare 2 independent groups and Kruskall Wallis test was used to compare more than 2 independent groups. DUNN test was used for post-hoc comparisons. Spearman correlation analysis was done to identify
Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
S. Tabur et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
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Table 1 Clinical and metabolic parameters of the microalbuminuric diabetic, normoalbuminuric diabetic and healthy control groups. Parameter
Microalbuminuric diabetic (n = 23)
Normoalbuminuric diabetic (n = 23)
Healthy control (n = 25)
p value
Age (years) Sex (F/M) BMI (kg/m2) Duration of DM (years) HbA1c (%) Insulin use SBP (mmHg) DBP (mmHg) FPG (mg/dl) LDL (mg/dl) HDL (mg/dl) BUN (mg/dl) Cr (mg/dl) Urinary microalbumin/Cr (mg/L) Serum U-II (ng/ml) TAS (μmol H2O2) TOS (mmol Equiv./l) OSI (arbitrary unit) PON-1 (U/ml) Arylesterase (U/ml)
50 ± 9a 12/11 27.43 ± 3.23a 4 ± 7a 8.50 ± 3.50a 7 120 ± 20a 80 ± 20a 168 ± 94a 110.17 ± 26.21 34.73 ± 6.47 31.17 ± 8.25 0.82 ± 0.30a 52 ± 68* 60 ± 26a,b,c 0.90 ± 0.16 19.05 ± 8.3a 2.27 ± 0.46 105 ± 122a 196.44 ± 27.87
46 ± 9a 13/10 27.28 ± 3.13a 4 ± 5a 8.80 ± 4.10a 2 120 ± 20a 80 ± 20a 170 ± 104a 107.95 ± 28.37 36.34 ± 6.04 26.65 ± 7.05 0.83 ± 0.20a 11 ± 9a 46 ± 22a,d 0.99 ± 0.11 15.03 ± 2.68a 1.65 ± 0.46 113 ± 101a 202.04 ± 38.94
47 ± 11.50a 15/10 26.70 ± 3.02a
0.270 0.863 0.292 0.095 0.767 0.066 0.677 0.648 0.0001 0.375 0.064 0.070 0.622 0.0001 0.0001 0.0001 0.0001 0.0001 0.005 0.016
120 ± 20a 75 ± 20a 87 ± 20a 119.08 ± 31.99 39.40 ± 7.85 27.16 ± 6.12 0.90 ± 0.10a 8 ± 11a 13 ± 6a 1.11 ± 0.12 15.44 ± 2.14a 1.38 ± 0.22 194 ± 139.5 223.99 ± 34.80
DM, diabetes mellitus; BMI, body mass index; FPG, fasting plasma glucose; HDL/LDL, high-density lipoprotein/low-density lipoprotein; BUN, blood urea nitrogen; Cr, creatinin; OSI, oksidative stress index; PON, paraoxonase; SBP/DBP, systolic blood pressure/diastolic blood pressure; TAS, total antioksidant status; TOS, total oxidative status. a Data in which non-parametric tests were used and expressed as median (Inter quarter range) p b 0.0001. b Microalbuminuric diabetic versus normalbuminurik diabetic; p b 0.0001. c Microalbuminuric diabetic versus control; p b 0.0001. d Normalbuminuric diabetic versus control; p b 0.0001.
associations between the parameters. Multiple linear regression analysis was conducted to identify the variables impacting the U-II level. Multiple logistic regression analysis was done to evaluate the relationship between microalbuminuria and TAS, TOS, PON, arylesterase, and U-II in diabetic patients. SPSS for Windows version 15 software was used for statistical analyses. The level of significance was set at p ≤ 0.05.
3. Results The mean age of all 3 groups and their gender distribution were similar (p = 0.270 and p = 0.863, respectively). BMI, SBP, DBP, LDL-C, HDL-C, blood urea nitrogen (BUN), and creatinine levels were not significantly different across the groups (p = 0.292, p = 0.677, p = 0.648, p = 0.375, p = 0.064, p = 0.070, and p = 0.622, respectively). The diabetic patient groups with and without microalbuminuria were not different in terms of duration of DM, insulin use, and HbA1c levels (p = 0.095, p = 0.066, and p = 0.767, respectively; Table 1). TAS level was lower in the microalbuminuric and normoalbuminuric diabetic groups compared to the healthy control group (p = 0.0001 and p = 0.006, respectively). While the TAS level was lower in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group, the difference was not significant (p = 0.093). OSI levels were higher in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group and the healthy control group (p = 0.0001 and p = 0.0001, respectively). The OSI levels in the normoalbuminuric group were higher than those in the healthy control group and the difference could not reach the level of significance (p = 0.054). TOS levels were higher in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group and the healthy control group (p = 0.002 and p = 0.0001, respectively). However, there was no significant difference between the normoalbuminuric group and the healthy control group in terms of TOS levels (p = 0.861). PON levels were lower in the microalbuminuric diabetic group and the normoalbuminuric diabetic group compared to the healthy control group (p = 0.005 and p = 0.005, respectively). The groups with and without microalbuminuria did not differ significantly with respect to PON levels (p = 0.930).
Serum U-II levels were higher in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group and the healthy control group (p = 0.009 and p = 0.0001, respectively). Normoalbuminuric diabetic group's serum U-II levels were significantly higher compared to the healthy control group (p = 0.0001; Fig. 1). Arylesterase levels were lower in the microalbuminuric diabetic group compared to the healthy control group (p = 0.021). On the other hand, there was no significant difference between diabetic groups with and without microalbuminuria or between the normoalbuminuric group and the healthy control group (p = 0.844 and p = 0.089, respectively). Correlation analysis yielded that serum U-II levels are weak negatively correlated to TAS, arylesterase, and PON levels (r = −0.395, p = 0.001; r = −0.291, p = 0.014; and r = −0.279, p = 0.018, respectively) and that they had a weak positive correlation with TOS levels (r = 0.312, p = 0.008; Figs. 2–5). These associations were confirmed in the multiple regression analysis (Table 2). Multiple logistic regression analysis results demonstrated the important association between microalbuminuria development and
Fig. 1. Serum concentrations of U-II in healthy volunteers and diabetic patients with or without microalbuminuria.
Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
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S. Tabur et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
Spearman correlation analysis (r=-0.395 , p= 0.001 )Total antioxidant status, TAS
Spearman correlation analysis (r=-0.279 , p= 0.018 ), Paraoxonase, PON
Fig. 2. Correlation between serum U-II levels and TAS. Spearman correlation analysis (r = −0.395, p = 0.001). Total antioxidant status, TAS.
Fig. 4. Correlation between serum U-II levels and PON. Spearman correlation analysis (r = −0.279, p = 0.018). Paraoxonase, PON.
oxidative stress and serum U-II levels in diabetic individuals (3).
the dilated tubular epithelium or damaged tubules. It has shown that the U-II has a direct role in tubular toxicity (Langham et al., 2004). A 24.3% decrease has been established in daily albumin excretion of diabetic nephropathy patients with macroalbuminuria by administration of urotensin receptor antagonist, palosuran (Sidharta et al., 2006). Similarly, in diabetic rats, palosuran has been demonstrated to prevent the progressive increase in albuminuria, renal dysfunction, and increases in tubular and tubulointerstitial lesions (Clozel, Hess, Qiu, Ding, & Rey, 2006). There is no study evaluating the effect of urotensin receptor antagonists in early diabetic nephropathy patients. Different mechanisms are activated as diabetic nephropathy progresses. We believe that treatment effectiveness could be higher in early stage. Various studies have shown that the oxidative stress develops due to oxidant and antioxidant balance impairments (Bedard & Krause, 2007). The role of oxidative stress in development of diabetic nephropathy has been demonstrated (King & Loeken, 2004). TAS, TOS, and OSI levels were analyzed as oxidative stress markers in this study. TAS level was lower in the microalbuminuric and normoalbuminuric diabetic groups compared to the healthy control group (p = 0.0001 and p = 0.005, respectively). TOS and OSI levels were higher in the microalbuminuric diabetic group compared to
4. Discussion This is the first study demonstrating the association between the increased serum U-II levels and early nephropathy in type 2 DM patients. In addition to serum U-II levels, increased oxidative stress is also an independent risk factor for development of early diabetic nephropathy. Similar to our results, Totsune et al. have also detected higher serum U-II levels in diabetics compared to healthy control patients. However, they did not detect a difference between the serum U-II levels of diabetic patients with and without proteinuria (Totsune et al., 2003). The difference between our results and theirs may be due to the low sample size (16 diabetic patients) of their study and that they had taken 100 mg albumin/g Cr as the cut-off point for proteinuria. In this case, patients with microalbuminuria are also placed in the group without proteinuria. In this study, we took 30–300 mg albumin/g Cr value as indication of microalbuminuria because microalbuminuria is the best indicator of early nephropathy. It has been shown that the expression of urotensin receptors in kidneys of diabetic patients is increased. This is observed especially in
Spearman correlation analysis (r= -0.291 , p= 0.014 )
Fig. 3. Correlation between serum U-II levels and arylesterase. Spearman correlation analysis (r = −0.291, p = 0.014).
Spearman correlation analysis (r=0.312 , p=0.008 ), TOS, total oxidant status
Fig. 5. Correlation between serum U-II levels and TOS. Spearman correlation analysis (r = 0.312, p = 0.008). TOS, total oxidant status.
Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
S. Tabur et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx Table 2 Multiple linear regression analysis between U-II and oxidant and antioxidant markers.
TAS TOS Arylesterase PON-1
β coefficients
p value
−0.879 1.296 −0.080 −0.027
0.016⁎ 0.016⁎ 0.451 0.035⁎
TAS, total antioxidant status; TOS, total oxidant status; PON, paraoxonase. ⁎ Significant at p b 0.05.
the normoalbuminuric diabetic group and the healthy control group (p = 0.002; p = 0.0001; p = 0.0001; p = 0.0001, respectively). Though the TOS and OSI levels were higher in the normoalbuminuric diabetic group compared to the healthy control group, the differences were not significant (p = 0.861 and p = 0.054, respectively). This shows that in the impairment in the balance of oxidants and antioxidants, the decrease in the antioxidants occurs prior to the increase in the oxidants. Continued oxidant level increase is thought to contribute to the progression into nephropathy. In our study, we evaluated the correlation between oxidative stress and serum U-II levels as well. There was a negative correlation between serum UII level and TAS and a positive correlation between TOS and OSI (p = 0.016 and p = 0.016, respectively). Based on the multiple regression analysis results, oxidative stress was shown to be an independent risk factor responsible for increases in the serum U-II levels (Table 3). PON-1 is an important molecule in atherosclerosis pathogenesis. Studies have determined lower PON1 activities in type 1 and 2 DM compared to healthy individuals (Ferretti, Bacchetti, Busni, Rabini, & Curatola, 2004; Flekac, Skrha, Zidková, Lacinová, & Hilgertová, 2008; Karabina, Lehner, Frank, Parthasarathy, & Santanam, 2005). In accordance with other studies, the PON1 level was lower in both of the diabetic groups with and without microalbuminuria compared to the healthy control group (p = 0.005 and p = 0.005, respectively). The role of PON1 activities in the development of DM-related complications has been investigated. A negative correlation was detected between the PON1 activities and presence of vascular complications (Karabina et al., 2005). Diabetic patients with neuropathy were demonstrated to have lower PON1 levels compared to diabetes patients without neuropathy (Poh & Muniandy, 2010). Another study has shown an association between PON1 expression and development of diabetic nephropathy in type 2 DM patients (Sun, 2010). In this study, the PON and arylesterase levels were lower in the microalbuminuric diabetic group compared to the normoalbuminuric diabetic group, but the difference was not significant (p = 0.930 and p = 0.844). Correlation analysis yielded that the U-II levels were negatively correlated to arylesterase and PON (r = − 0.291, p = 0.014; r = − 0.279, p = 0.018, respectively). Multiple regression analysis has shown that decreasing levels of PON is an independent risk factor responsible for increases in the serum U-II levels (p = 0.035). Consequently, U-II system has a role in the development of early diabetic nephropathy in type 2 DM. U-II system is affected by oxidative stress. Further studies on this topic will help to better
Table 3 Results of logistic regression models predicting microalbuminuria.
TAS TOS Arylesterase PON-1
OR (95% CI)
p value
0.001 1.419 1.005 0.991
0.011⁎ 0.007⁎ 0.720 0.161
(0.0001–0.134) (1.098–1.834) (0.980–1.030) (0.979–1003)
TOS, total oxidant status; PON, paraoxonase. ⁎ Significant at p b 0.05, TAS, total antioxidant status.
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Please cite this article as: Tabur, S., et al., Urotensin-II level and its association with oxidative stress in early diabetic nephropathy, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.07.011
